Swellable synthetic microcarriers for culturing cells

ABSTRACT

A cell culture microcarrier includes a polymer formed from copolymerization of a mixture including (i) an uncharged hydrophilic unsaturated monomer having a hydroxyl group; (ii) a hydrophilic carboxylic acid containing unsaturated monomer; and (iii) a hydrophilic multifunctional unsaturated monomer. The microcarrier may further include a polypeptide, such as a polypeptide that promotes cell adhesion, conjugated to the surface of the microcarrier; e.g. via the carboxyl group from the hydrophilic carboxylic acid containing unsaturated monomer. Some of the microcarriers support attachment of human embryonic stem cells.

CLAIMING BENEFIT OF PRIOR FILED U.S. APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/181,776 filed on May 28, 2009. The content of this document andthe entire disclosure of publications, patents, and patent documentsmentioned herein are incorporated by reference.

FIELD

The present disclosure relates to cell culture microcarriers, and moreparticularly to synthetic, chemically-defined microcarriers.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as textfiled named “SP10173_ST25.txt” having a size of 8 kb and created on May27, 2010. Due to the electronic filing of the Sequence Listing, theelectronically submitted Sequence Listing serves as both the paper copyrequired by 37 CFR §1.821(c) and the CRF required by §1.821(e). Theinformation contained in the Sequence Listing is hereby incorporatedherein by reference.

BACKGROUND

Microcarriers have been employed in cell culture for the purpose ofproviding high yields of attachment-dependent cells. Microcarriers aretypically stirred or agitated in cell culture media and provide a verylarge attachment and growth surface area to volume ratio relative tomore traditional culture equipment.

Most currently available microcarriers provide for non-specificattachment of cells to the carriers for cell growth. While useful, suchmicrocarriers do not allow for biospecific cell adhesion and thus do notreadily allow for tailoring of characteristics of the cultured cells.For example, due to non-specific interactions it may be difficult tomaintain cells, such as stem cells, in a particular state ofdifferentiation or to direct cells to differentiate in a particularmanner.

Some currently available microcarriers provide for bio-specificadhesion, but employ animal derived coating such as collagen or gelatin.Such animal derived coatings can expose the cells to potentially harmfulviruses or other infectious agents which could be transferred topatients if the cells are used for a therapeutic purpose. In addition,such viruses or other infectious agents may compromise general cultureand maintenance of the cultured cells. Further, such biological productstend to be vulnerable to batch variation and limited shelf-life.

BRIEF SUMMARY

Among other things, the present disclosure described synthetic,chemically-defined microcarriers useful in culturing cells. Themicrocarriers described herein are, in various embodiments, durableenough to withstand stirring while maintaining cell adhesion and growth,even though the microspheres may be highly swellable.

In various embodiments, a microcarrier includes a polymer formed fromcopolymerization of a mixture including (i) an uncharged hydrophilicunsaturated monomer having a hydroxyl group; (ii) a hydrophiliccarboxylic acid containing unsaturated monomer; and (iii) a hydrophilicmultifunctional unsaturated monomer. The microcarrier may furtherinclude a polypeptide, such as a polypeptide that promotes celladhesion, conjugated to the surface of the microcarrier; e.g. via thecarboxyl group from the hydrophilic carboxylic acid containingunsaturated monomer. Preferably, the polymeric base does not allow fornon-specific adhesion of cells, and the polypeptide provides forbio-specific cell binding.

In various embodiments, a method for producing a cell culturemicrocarrier includes copolymerizing a mixture of monomers to form themicrocarrier base. The mixture of monomers includes (i) an unchargedhydrophilic unsaturated monomer having a hydroxyl group; (ii) ahydrophilic carboxylic acid containing unsaturated monomer; and (iii) ahydrophilic multifunctional unsaturated monomer. In some embodiments,the mixture of monomers is copolymerized by water-in-oilcopolymerization. The method further includes conjugating a polypeptideto the microcarrier base to form the microcarrier.

In numerous embodiments, a method for culturing cells includescontacting the cells with a cell culture medium having microcarriers.The microcarriers include a polymeric base formed from a mixture ofmonomers including (i) an uncharged hydrophilic unsaturated monomerhaving a hydroxyl group; (ii) a hydrophilic carboxylic acid containingunsaturated monomer; and (iii) a hydrophilic multifunctional unsaturatedmonomer; and include a polypeptide conjugated polymer. The methodfurther includes culturing the cells in the medium. The cells may bestem cells, such as embryonic stem cells, and the medium may be achemically defined medium.

One or more of the various embodiments presented herein provide one ormore advantages over prior articles and systems for culturing cells. Forexample, synthetic microcarriers described herein have been shown tosupport cell adhesion without the need of animal derived biocoatingwhich limits the risk of pathogen contamination. This is especiallyrelevant when cells are dedicated to cell therapies. Further, largescale culture of cells, including human embryonic stem cells (hESCs), ispossible with microcarriers as described herein. Such microcarriers mayalso be advantageously used for culturing cells other than stem cellswhen the presence of animal derived products such as collagen, gelatin,fibronectin, etc. are undesired or prohibited. The methods describedherein allow for the preparation of microcarriers having a wide range ofproperties such as stiffness, swellability, surface chemistries, and canprovide microcarriers having a soft swellable substrate that preventscell damage when cells are cultured in stirred tanks. Further, themicrocarriers may be monolithic and not coated as most of the commercialmicrocarriers, reducing the number of components to worry about withregard to manufacturing complexity and cell compatibility. These andother advantages will be readily understood from the following detaileddescriptions when read in conjunction with the accompanying drawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is microscopic image of representative microcarriers preparedaccording to Example 1.

FIGS. 2 A and B are is scanning electron microscope (SEM) images ofrepresentative microcarriers prepared according to Example 1.

FIG. 3A-D are SEM images of representative microspheres obtained afterPBS buffer washing at different magnifications.

FIG. 4 is a graph of the size distribution of microcarrier particlesprepared according to Example 1.

FIGS. 5A and B are is images of CHO-M1 cells adhering on microcarriersgrafted with GRGDS peptide.

FIG. 6 is an image of HEK293 cells adhering on microcarriers graftedwith GRGDS peptide.

FIG. 7 is an image of MRC5 cells adhering on microcarriers grafted withGRGDS peptide

FIG. 8 is a microscopic image of representative microcarriers preparedaccording to Example 6.

FIGS. 9A and B are is a phase contrast microscopy image of HT1080 cellsadhered to a vitronectin polypeptide (A) and an RGE polypeptide (B)grafted to microcarriers, as discussed in Examples 7 and 8,respectively.

FIG. 10 is a graph showing the size of microcarriers obtained as afunction of stirring rate.

FIG. 11 is a phase contrast microscopy image illustrating HT1080adhesion after 2 hours incubation on microcarriers.

FIGS. 12A-D are a series of phase contrast microscopy image illustratingHT1080 cell adhesion and growth after 2 hours and 4 days in spinnerflask on VN grafted microcarriers (FIGS. 12 A and B) prepared inaccordance with the teachings presented herein or on comparativeCytodex3 microcarriers (FIGS. 12C and D).

FIG. 13 is a graph of cell growth on different types of microcarriers,as well as reported growth on commercial Solohill pronectinFmicrocarriers. For all conditions cells were grown in spinner flask for4 days under intermittent stirring.

FIG. 14 is a phase contrast microscopy image of ES-D3 mouse pluripotentembryonic stem cells adhesion and growth after 48 hours on peptidegrafted microcarriers in serum free conditions and in the presence of 10μM of Y27632 ROCK kinase inhibitor.

FIG. 15 is a graph showing the alkaline phosphatase activity in cellextracts performed at the end of the experiment in Example 15, where STOmouse fibroblasts were used as a negative control.

FIG. 16 is a phase contrast microscopy image showing ES-D3 mouseembryonic stem cells adhering on the microcarriers of Example 14(μHG14). The cells were incubated for 18 hours with the beads in mTeSR1serum free medium

FIG. 17 is a microscopy image illustrating BG01V cells attachment onpeptide grafted microcarriers 5 days after seeding.

FIG. 18 is a graph presenting the result of BG01V cell growth in stiffedconditions on different types of microcarriers. Matrigel (Mg) coatedglass carriers are used as a gold standard, non coated (KO) glass beadsare used as a negative control.

FIG. 19 is a graph illustrating the relationship between VN peptideloading (in nmol/mg) vs weight percent of crosslinker.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings that form a part hereof, and in which are shown byway of illustration several specific embodiments of devices, systems andmethods. It is to be understood that other embodiments are contemplatedand may be made without departing from the scope or spirit of thepresent disclosure. The following detailed description, therefore, isnot to be taken in a limiting sense.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise. As used inthis specification and the appended claims, the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

Polypeptide sequences are referred to herein by their one letter aminoacid codes and by their three letter amino acid codes. These codes maybe used interchangeably.

As used herein, “monomer” means a compound capable of polymerizing withanother monomer, (regardless of whether the “monomer” is of the same ordifferent compound than the other monomer), which compound has amolecular weight of less that about 1000 Dalton. In many cases, monomerswill have a molecular weight of less than about 400 Dalton.

As used herein, “microcarrier base” means a polymeric microcarrier onwhich a polypeptide may be conjugated. “Microcarrier base” and“polymeric microcarrier” are often used herein interchangeably. Amicrocarrer is small discrete particle for use in culturing cells and towhich cells may attach. Microcarriers may be in any suitable shape, suchas rods, spheres, and the like, and may be porous or non-porous.

As used herein “peptide” and “polypeptide” mean a sequence of aminoacids that may be chemically synthesized or may be recombinantlyderived, but that are not isolated as entire proteins from animalsources. For the purposes of this disclosure, peptides and polypeptidesare not whole proteins. Peptides and polypeptides may include amino acidsequences that are fragments of proteins. For example peptides andpolypeptides may include sequences known as cell adhesion sequences suchas RGD. Polypeptides may be of any suitable length, such as betweenthree and thirty amino acids in length. Polypeptides may be acetylated(e.g. Ac-LysGlyGly) or amidated (e.g. SerLysSer-NH₂) to protect themfrom being broken down by, for example, exopeptidases. It will beunderstood that these modifications are contemplated when a sequence isdisclosed.

As used herein, a “(meth)acrylate monomer” means a methacrylate monomeror an acrylate monomer. As used herein “(meth)acrylamide monomer” meansa methacrylamide or an acrylamide monomer. (Meth)acrylate and(meth)acrylamide monomers have at least one ethylenically unsaturatedmoiety. “Poly(meth)acrylate”, as used herein, means a polymer formedfrom one or more monomers including at least one (meth)acrylate monomer.“Poly(meth)acrylamide”, as used herein, means a polymer formed from oneor more monomers including at least one (meth)acrylamide monomer.

As used herein, “equilibrium water content” refers to water-absorbingcharacteristic of a polymeric material and is defined and measured byequilibrium water content (EWC) as shown by Formula 1:

EWC (%)=[(Wgel−Wdry)/(Wgel)]*100.  Formula 1

As used herein, “have”, “having”, “include”, “including”, “comprise”,“comprising” or the like are used in their open ended sense, andgenerally mean “including, but not limited to”. It will be understoodthat “consisting essentially of”, “consisting of”, and the like aresubsumed in “comprising” and the like. Accordingly, a microcarrier baseformed from a mixture of monomers comprising an uncharged hydrophilicunsaturated monomer having a hydroxyl group; a hydrophilic carboxylicacid containing unsaturated monomer; and a hydrophilic multifunctionalunsaturated monomer may be formed from a mixture consisting essentiallyof, or consisting of, an uncharged hydrophilic unsaturated monomerhaving a hydroxyl group; a hydrophilic carboxylic acid containingunsaturated monomer; and a hydrophilic multifunctional unsaturatedmonomer.

As used herein, “hydrophilic”, as it relates to a monomer, means themonomer separates into the water phase of an oil-in-water emulsion. Forexample, 95% or more; e.g. 98% or more, of the monomer separates intothe water phase. It will be understood that the amount of monomer thatwill remain in the water phase depends on the components of the emulsion(e.g., components of the oil phase and emulsifier, if any). For example,in many emulsions where the oil phase is silicone oil or a fluorinatedsolvent, monomers that are not typically considered very hydrophilic,such as ethylene glycol dimethacrylate, may remain dispersed in thewater phase (and thus would be considered “hydrophilic” herein). Theability of a monomer to remain in the water phase in an oil-in-wateremulsion is important when microcarriers are formed via oil-in-watercopolymerization. If the monomer does not remain in the water phase, theability to form microcarriers may be compromised. Accordingly, in manyembodiments, the monomers are at least water-miscible, and arepreferably water soluble, to form at least 5 weight percent solutionswhen dissolved in water. In some embodiments, hydrophilic monomers havean octanol/water partition coefficient of less than 1.9, less than 1.8,less than 1.7, less that 1.6, less than 1.5, or less than 1.4.

The present disclosure describes, inter alia, synthetic microcarriersfor culturing cells. In various embodiments, the microcarriers areconfigured to support proliferation and maintenance of undifferentiatedstem cells in chemically defined media.

1. Microcarrier

A microcarrier, as described herein, is formed by polymerization of amixture of monomers including an uncharged hydrophilic unsaturatedmonomer having a hydroxyl group, a hydrophilic carboxylic acidcontaining unsaturated monomer, and a hydrophilic multifunctionalunsaturated monomer. In some embodiments, the polymeric base of themicrocarrier is formed from a mixture of monomers consisting of, orconsisting essentially of an uncharged hydrophilic unsaturated monomerhaving a hydroxyl group, a hydrophilic carboxylic acid containingunsaturated monomer, and a hydrophilic multifunctional unsaturatedmonomer.

A. Uncharged Hydrophilic Unsaturated Monomer Having a Hydroxyl Group

Any suitable uncharged hydrophilic unsaturated monomer having a hydroxylgroup may be employed. An “uncharged” monomer is a monomer that, whenincorporated into a polymeric microcarrier, is free of charged groupsunder a given cell culture condition. Microcarriers having chargedmoieties under cell culture conditions can result in non-specificattachment of cells. It is desired, in various embodiments, for cellinteraction with a microcarrier to be biospecific and selective to apolypeptide grafted to the microcarrier.

In various embodiments, the uncharged hydrophilic unsaturated monomerhaving a hydroxyl group is a (meth)acrylate monomer of Formula (I):

where A is H or methyl, and where B is C1-C6 straight or branched chainalcohol or ether. In some embodiments, B is C1-C4 straight or branchedchain alcohol. By way of example, hydroxypropyl methacrylate,2-hydroxyethyl methacrylate (HEMA), 2-hydroxyethyl acrylate, glycerolmethacrylate, hydroxypropyl acrylate, 4-hydroxybutyl acrylate, or thelike may be employed.

In various embodiments, the uncharged hydrophilic unsaturated monomerhaving a hydroxyl group is a (meth)acrylamide monomer of Formula (II):

where A is hydrogen or methyl, and where B is C1-C6 straight or branchedchain alcohol or ether. In some embodiments, B is C1-C4 straight orbranched chain alcohol. For example, the uncharged hydrophilicunsaturated monomer may be N-(hydroxymethyl)acrylamide,N-[Tris(hydroxymethyl)methyl]acrylamide, 3-acryloylamino-1-propanol,N-acrylamido-ethoxyethanol, N-hydroxyethyl acrylamide, or the like.

B. Hydrophilic Carboxylic Acid Containing Unsaturated Monomer

Any suitable hydrophilic carboxylic acid containing unsaturated monomermay be employed. In various embodiments, the hydrophilic carboxylic acidcontaining unsaturated monomer is a (meth)acrylate monomer of Formula(III):

where A is hydrogen or methyl, and where D is C1-C6 straight or branchedchain alkyl substituted with a carboxyl group (—COOH). In someembodiments, D is straight or branched chain C1-C3 substituted with acarboxyl group. By way of example, the hydrophilic carboxylic acidcontaining unsaturated monomer may be 2-carboxyethyl methacrylate,2-carboxyethyl acrylate, acrylic acid, methacrylic acid or the like.

In various embodiments, the hydrophilic carboxylic acid containingunsaturated monomer is a (meth)acrylamide monomer of Formula (IV):

where A is hydrogen or methyl, and where D is C1-C6 straight or branchedchain alkyl substituted with a carboxyl group (—COOH). In someembodiments, D is straight or branched chain C1-C3 substituted with acarboxyl group. By way of example, the hydrophilic carboxylic acidcontaining unsaturated monomer may be 2-carboxyethyl acrylamide,acrylamidoglycolic acid, or the like.

C. Hydrophilic Multifunctional Unsaturated Monomer

Any suitable and a hydrophilic multifunctional unsaturated monomer maybe employed. As used herein, “multifunctional monomer” means a monomerhaving more than one group capable of polymerizing. Multifunctionalmonomers can serve as cross-linking agents. Multifunctional monomers maybe di-, tri-, or higher functions. In various embodiments,multifunctional monomers are difunctional. Multifunctional monomers mayhave any suitable polymerizable group. In various embodiments,multifunctional monomers have a vinyl group; e.g. a (meth)acrylate groupor a (meth)acrylamide group. Examples of suitable multifunctionalmonomers include N,N′ methylenebisacrylamide,N,N′(1,2-dihydroxyethylene)bisacrylamide, polyethylene glycoldi(meth)acrylate, triglycerol diacrylate, propylene glycol glycerolatediacrylate, trimethylolpropane ethoxylate triacrylate, glycerol1,3-diglycerolate diacrylate, and the like.

In various embodiments, the hydrophilic multifunctional unsaturatedmonomer is more hydrophilic than tetra(ethylene glycol) dimethacrylate.For example, the hydrophilic multifunctional unsaturated monomer mayhave an octanol/water partition coefficient that is less than theoctanol/water coefficient of tetra(ethylene glycol) dimethacrylate. Insome embodiments, hydrophilic monomers have an octanol/water partitioncoefficient of less than 1.9, less than 1.8, less than 1.7, or less that1.6. In many embodiments, the monomers are at least water-miscible, andare preferably water soluble, to form at least 5 weight percentsolutions when dissolved in water.

However, in some embodiments, a hydrophobic or less hydrophilicunsaturated multifunctional monomer is used in addition to thehydrophilic multifunctional unsaturated monomer. For example,tetra(ethylene glycol) dimethacrylate may be employed in addition to ahydrophilic multifunctional unsaturated monomer, such as methylenebisacrylamide (see, e.g., EXAMPLE 17).

D. Formation of Polymeric Microcarrier Base

A microcarrier may be formed via any suitable polymerization reaction ofthe mixture of monomers. Any suitable amount of an uncharged hydrophilicunsaturated monomer having a hydroxyl group, a hydrophilic carboxylicacid containing unsaturated monomer, and a hydrophilic multifunctionalunsaturated monomer may be employed in the mixture. In variousembodiments, the mixture of monomers used to form the microcarrierincludes (i) about 30 to about 70 parts per weight of the unchargedhydrophilic unsaturated monomer having a hydroxyl group; (ii) about 20to about 60 parts per weight of the hydrophilic carboxylic acidcontaining unsaturated monomer; and (iii) 1 to 15 parts by weight of thehydrophilic multifunctional unsaturated monomer. Or, in embodiments, themixture of monomers used to form the microcarrier includes (i) about 30to about 70, about 30 to about 60, about 30 to about 55, about 30 toabout 50, about 30 to about 45 or about 30 to about 40 parts per weightof the uncharged hydrophilic unsaturated monomer having a hydroxylgroup; (ii) more than 20, about 20 to about 60, about 30 to about 60,about 35 to about 60, about 40 to about 60, about 45 to about 60 orabout 50 to about 60 per weight of the hydrophilic carboxylic acidcontaining unsaturated monomer; and (iii) about 1 to 15 or about 1 toabout 10 parts by weight of the hydrophilic multifunctional unsaturatedmonomer. Although the word “about” is used to describe these ranges, itwill be understood that these ranges are defined, and can be understoodto include or to not include the word “about”. As discussed above, ahydrophilic multifunctional unsaturated monomer (e.g., methylenebisacrylamide or dihydroethylene bisacrylamide) may be used incombination with a less hydrophilic or hydrophobic unsaturated monomer(e.g., tetra(ethylene glycol) dimethacrylate). In many of suchembodiments, the total crosslinker (multifunctional unsaturated monomer)does not exceed 15 parts by weight of the mixture of monomers, with themore hydrophilic multifunctional unsaturated monomer being at least 1%by weight of the mixture of monomers. The weight percentages ofmultifunctional unsaturated monomer are particularly well suited fordifunctional monomers. It will be understood that the ratio ofcrosslinker employed may be readily changed (e.g., reduced) if tri- orhigher functional monomers are used as crosslinkers.

The ratio of cross-linker may be changed. For example, as shown in FIG.19, the percentage of cross-linker employed may affect the peptideloading on the microcarrier. For example, a higher cross-linker weightpercent is correlated with a lower loading on the microcarriers. Inaddition, the cross-linker ratio may also affect the EWC of themicrocarrier.

In various embodiments, the pendant carboxyl content (from thehydrophilic carboxylic acid containing unsaturated monomer) of thepolymeric microcarrier is greater than about 1 milliequivalents pergram, greater than about 2 milliequivalents per gram, greater than about3 milliequivalents per gram, or about 4 milliequivalents per gram. Invarious embodiments, the cross-linking density (from the hydrophilicmultifunctional unsaturated monomer) of the polymeric microcarrier isbetween about 1×10⁻⁴ and 5×10⁻³ moles per gram, between about 1.5×10⁻³and 2.5×10⁻³ moles per gram, or about 1.7×10⁻³ moles per gram.

It will be understood that the relative amounts of the monomers and theproperties of the monomers will affect the desired properties of theresulting polymeric microcarrier. For example, it will be understoodthat the equilibrium water content (EWC) of the polymeric microcarriermay be controlled by the monomers chosen to form the microcarrier. Forexample, a higher degree of hydrophilicity of the monomers used, thehigher the EWC of the polymeric microcarrier will be. However, this maybe attenuated by increasing the percentage, or increasing thefunctionality, of the cross-linking monomer (the hydrophilicmultifunctional unsaturated monomer), which should reduce the ability ofthe SA layer to swell and thereby reduce the EWC. While not intending tobe bound by theory, it is believed that the EWC of the polymericmicrocarrier may be an important variable in determining what types ofcells the microcarrier can support in culture. The stiffness andswelling power of the microcarrier may mimic environments in whichcertain cells grow well. As presented in co-pending patent applications,U.S. patent application Ser. Nos. 12/362,924 and 12/362,974, swellablesurface layers having an EWC of between about 5% and about 70% weresuitable for culturing human embryonic stem cells in an undifferentiatedstate for at least five passages. In these copending applications, theswellable (meth)acrylate layers were formed in situ on a substrate. Ifthe EWC were too high, the layer may swell too much when exposed to anaqueous environment such as cell culture and delaminate. However, as themicrocarriers described herein are not formed as a layer on a substrate,delamination is not a concern and the EWC can be higher. In manyrespects, microcarriers with higher EWCs are desirable because they tendto have low stiffness and thus more closely resemble biological tissuestiffness. They are “soft” surfaces. They may also have hightransparency due to the high amount of water, and prevent or reduce celldamage during collision in stirred cultures. However, if themicrocarriers are too soft, they may be difficult to handle or mayaggregate and thus may be difficult to disperse in culture medium. Byadjusting the crosslinker percentage, and the chemical nature of thecrosslinker, the EWC can be “tuned.”

While microcarriers as described herein can have any suitable EWC, invarious embodiments, a microcarrier as described herein has an EWC ofgreater than about 60%, greater than about 65%, greater than about 70%,greater than about 75%, greater than about 80%, greater than about 85%,or greater than about 90%. Due in part to the use of a carboxylcontaining monomer in the microcarriers of various embodiments describedherein, the EWC may be pH dependent. For example, the EWC of particularmicrocarriers may be higher in phosphate buffer (pH 7.4) than indistilled, deionized water (pH ˜5). In various embodiments, the EWC of amicrocarrier in distilled, deionized water is greater than about 60%,greater than about 65%, greater than about 70%, greater than about 75%,greater than about 80%, greater than about 85%, or greater than about90%

As discussed further below, one or more polypeptides may be conjugatedto microcarrier, which may affect the EWC of the microcarrier (typicallyincreasing the EWC). The amount of polypeptide conjugated to amicrocarrier tends to be variable and can change depending on the size(e.g., diameter) of the microcarrier. Accordingly, the EWC ofmicrocarrier with conjugated polypeptide prepared in accordance with astandard protocol may be variable. For purposes of reproducibility, itmay be desirable to measure the EWC of microcarriers prior toconjugation with a polypeptide. With this noted, in some embodiments,after the microcarriers have been conjugated with polypeptides, the EWCof embodiments of microcarrier-polypeptide conjugates may be greaterthan about 50%, greater than about 55%, greater than about 60%, greaterthan about 65%, greater than about 70%, greater than about 75%, greaterthan about 80%, greater than about 85%, or greater than about 90% inwater.

Once the appropriate monomers in the appropriate amounts are selected,the polymeric microcarrier may be formed via polymerization reaction. Inaddition to the monomers that form the microcarrier, a composition forforming the microcarrier may include one or more additional compoundssuch as surfactants, wetting agents, photoinitiators, thermalinitiators, catalysts, and activators.

Any suitable polymerization initiator may be employed. One of skill inthe art will readily be able to select a suitable initiator, e.g. aradical initiator or a cationic initiator, suitable for use with themonomers. In various embodiments, UV light is used to generate freeradical monomers to initiate chain polymerization. Examples ofpolymerization initiators include organic peroxides, azo compounds,quinones, nitroso compounds, acyl halides, hydrazones, mercaptocompounds, pyrylium compounds, imidazoles, chlorotriazines, benzoin,benzoin alkyl ethers, diketones, phenones, or mixtures thereof. Examplesof suitable commercially available, ultraviolet-activated and visiblelight-activated photoinitiators have tradenames such as IRGACURE 651,IRGACURE 184, IRGACURE 369, IRGACURE 819, DAROCUR 4265 and DAROCUR 1173commercially available from Ciba Specialty Chemicals, Tarrytown, N.Y.and LUCIRIN TPO and LUCIRIN TPO-L commercially available from BASF(Charlotte, N.C.)

A photosensitizer may also be included in a suitable initiator system.Representative photosensitizers have carbonyl groups or tertiary aminogroups or mixtures thereof. Photo sensitizers having a carbonyl groupsinclude benzophenone, acetophenone, benzil, benzaldehyde,o-chlorobenzaldehyde, xanthone, thioxanthone, 9,10-anthraquinone, andother aromatic ketones. Photo sensitizers having tertiary amines includemethyldiethanolamine, ethyldiethanolamine, triethanolamine,phenylmethyl-ethanolamine, and dimethylaminoethylbenzoate. Commerciallyavailable photo sensitizers include QUANTICURE ITX, QUANTICURE QTX,QUANTICURE PTX, QUANTICURE EPD from Biddle Sawyer Corp.

In general, the amount of photosensitizer or photoinitiator system mayvary from about 0.01 to 10% by weight.

Examples of cationic initiators that may be employed include salts ofonium cations, such as arylsulfonium salts, as well as organometallicsalts such as ion arene systems.

Examples of free radical initiators that may be employed includeazo-type initiators such as 2-2′-azobis(dimethyl-valeronitrile),azobis(isobutyronitrile), azobis(cyclohexane-nitrite),azobis(methyl-butyronitrile), and the like, peroxide initiators such asbenzoyl peroxide, lauroyl peroxide, methyl ethyl ketone peroxide,isopropyl peroxy-carbonate,2,5-dienethyl-2,5-bas(2-ethylhexanoyl-peroxy)hexane, di-tert-butylperoxide, cumene hydroperoxide, dichlorobenzoyl peroxide, potassiumpersulfate, ammonium persulfate, sodium bisulfate, combination ofpotassium persulfate, sodium bisulfate and the like, and mixturesthereof. Of course, any other suitable free radical initiators may beemployed. An effective quantity of an initiator is generally within therange of from about 0.1 percent to about 15 percent by weight of thereaction mixture, such as from 0.1 percent to about 10 percent by weightor from about 0.1 percent to about 8 percent by weight of the reactionmixture.

In various embodiments, one or more monomers are diluted with waterprior to undergoing polymerization.

(Meth)acrylate monomers, (meth)acrylamide monomers, or other suitablemonomers may be synthesized as known in the art or obtained from acommercial vendor, such as Polysciences, Inc., Sigma Aldrich, Inc., andSartomer, Inc.

E. Water-in-Oil Emulsion Copolymerization

Microcarriers may be formed in any suitable manner. It will beunderstood that the size and shape of the resulting polymericmicrocarriers will be affected by the reaction conditions employed. Innumerous embodiments, water-in-oil copolymerization is employed to formspherical microcarriers. Using such a reaction scheme and system,hydrophilic monomers can be dissolved in water and added to an oil orhydrophobic solution or suspension. Any suitable hydrophobic liquid,such as octanol, toluene, alkane, such as heptane, hexane or higheralkane including decane, dodecane, hexadecane, heavy mineral oil,silicone oil, fluorinated solvent, or the like may be used. Anemulsifier may be added to promote the formation of the water-in-oilemulsion. Water insoluble (oil soluble) emulsifiers are preferred suchas those having HLB value (hydrophilic lipophilic balance) lower than 9.Examples of such emulsifiers are Sorbitan trioleate (Span 85) HLB=1.8,Sorbitan tristearate (Span 65) HLB=2.1, Sorbitan sesquioleate (Arlacel83) HLB=3.7, Glyceryl monostearate, HLB=3.8, Sorbitan monooleate, (Span80) HLB=4.3, Sorbitan monostearate, (Span 60) HLB=4.7, Sorbitanmonopalmitate, (Span 40) HLB=6.7 Sorbitan monolaurate, (Span 20)HLB=8.6. Hydrophobically modified water soluble polymers or randomcopolymers or block or graft copolymer which are built of components ofdifferent polarity are further examples of suitable emulsifiers. Oneexample of a hydrophobically modified water soluble polymer emulsifieris ethyl cellulose. Of course other such emulsifiers may be employed.

Regardless of how microcarriers are prepared, in embodiments, thatmicrocarriers have a density slightly greater than the cell culturemedium in which they are to be suspended to facilitate separation of themicrocarriers from the surrounding medium. In various embodiments, themicrocarriers have a density of about 1.01 to 1.10 grams per cubiccentimeter. Microcarriers having such a density should be readilymaintained in suspension in cell culture medium with gentle stirring. Itis expected that the density of the microcarriers can be easily tuned byvarying the water to monomer ratio.

In addition, it is preferred that the size variation of themicrocarriers is sufficiently small to ensure that most, if not all, ofthe microcarriers can be suspended with gentle stirring. By way ofexample, the geometric size distribution of the microcarriers may bebetween about 1 and 1.4. Microcarriers may be of any suitable size. Forexample, microcarriers may have a diametric dimension of between about20 microns and 1000 microns. Spherical microcarriers having suchdiameters can support the attachment of several hundred to thousands ofcells per microcarrier. The size of the microcarriers formed viawater-in-oil copolymerization techniques can be easily tuned by varyingthe stirring speed or the type of emulsifier used. For example, higherstirring speeds tend to result in smaller particle size (see, e.g., FIG.10). In addition, it is believed that the use of polymeric emulsifiers,such as ethylcellulose, enables larger particles relative to lowermolecular weight emulsifiers. Accordingly, one can readily modifystirring speed or agitation intensity and emulsifier to obtainmicrocarriers of a desired particle size.

It has been found that water-in-oil emulsion copolymerization can resultin spherical microcarriers that are non-porous. As used herein,“non-porous” means having no pores or pores of an average size smallerthan a cell with which the microcarrier is cultured, e.g., less thanabout 0.5-1 micrometers. Non-porous microspheres are desired when themicrocarriers are not degradable, because cells that enter pores ofmacroporous microcarriers are difficult to remove. However, if themicrocarriers are degradable, e.g. if they include an enzymatically orotherwise degradable cross-linker, it may be desirable for themicrocarriers to be macroporous.

In some embodiments, microcarriers are optically transparent, allowingfor easy microscopic observation of interaction of cells with amicrocarrier. A transparent microcarrier allows for the observation of acell on an opposing side (relative from the side from which it isviewed) of the microcarrier. The more transparent the microcarrier, themore readily the interaction can be observed via microscope.Microcarriers formed from 2-hydroxyethyl methacrylate, 2-carboxyethylacrylate and dihydroxyethylene bisacrylamide were found to be highlytransparent (see, e.g., Example 6 and FIG. 8). Microcarriers formed fromother (i) uncharged hydrophilic unsaturated monomers having a hydroxylgroup, (ii) hydrophilic carboxylic acid containing unsaturated monomers,and (iii) hydrophilic multifunctional unsaturated monomers should alsobe highly transparent, particularly when the monomers are (meth)acrylateor (meth)acrylamide monomers.

While not intending to be bound by theory, it is believed that moreswellable microcarriers or microcarriers having high EWCs tend to formtransparent microcarriers due to the high amount of water they absorb inculture media.

F. Conjugation of Polypeptide to Polymeric Microcarrier

Any suitable polypeptide may be conjugated to a microcarrier.Preferably, polypeptide includes an amino acid capable of conjugating tomicrocarrier; e.g. via the free carboxyl group formed from thehydrophyllic carboxylic acid containing unsaturated monomer. By way ofexample, any native or biomimetic amino acid having functionality thatenables nucleophilic addition; e.g. via amide bond formation, may beincluded in polypeptide for purposes of conjugating to the microcarrier.Lysine, homolysine, ornithine, diaminoproprionic acid, anddiaminobutanoic acid are examples of amino acids having suitableproperties for conjugation to a carboxyl group of the microcarrier. Inaddition, the N-terminal alpha amine of a polypeptide may be used toconjugate to the carboxyl group, if the N-terminal amine is not capped.In various embodiments, the amino acid of polypeptide that conjugateswith the microcarrier is at the carboxy terminal position or the aminoterminal position of the polypeptide.

In numerous embodiments, the polypeptide, or a portion thereof, has celladhesive activity; i.e., when the polypeptide is conjugated to themicrocarrier, the polypeptide allows a cell to adhere to the surface ofthe peptide-containing microcarrier. By way of example, the polypeptidemay include an amino sequence, or a cell adhesive portion thereof,recognized by proteins from the integrin family or leading to aninteraction with cellular molecules able to sustain cell adhesion. Forexample, the polypeptide may include an amino acid sequence derived fromcollagen, keratin, gelatin, fibronectin, vitronectin, laminin, bonesialoprotein (BSP), or the like, or portions thereof. In variousembodiments, polypeptide includes an amino acid sequence of ArgGlyAsp(RGD).

Microcarriers as described herein provide a synthetic surface to whichany suitable adhesion polypeptide or combinations of polypeptides may beconjugated, providing an alternative to biological substrates or serumthat have unknown components. In current cell culture practice, it isknown that some cell types require the presence of a biologicalpolypeptide or combination of peptides on the culture surface for thecells to adhere to the surface and be sustainably cultured. For example,HepG2/C3A hepatocyte cells can attach to plastic culture ware in thepresence of serum. It is also known that serum can provide polypeptidesthat can adhere to plastic culture ware to provide a surface to whichcertain cells can attach. However, biologically-derived substrates andserum contain unknown components. For cells where the particularcomponent or combination of components (peptides) of serum orbiologically-derived substrates that cause cell attachment are known,those known polypeptides can be synthesized and applied to amicrocarrier as described herein to allow the cells to be cultured on asynthetic surface having no or very few components of unknown origin orcomposition.

For any of the polypeptides discussed herein, it will be understood thata conservative amino acid may be substituted for a specificallyidentified or known amino acid. A “conservative amino acid”, as usedherein, refers to an amino acid that is functionally similar to a secondamino acid. Such amino acids may be substituted for each other in apolypeptide with a minimal disturbance to the structure or function ofthe polypeptide according to well known techniques. The following fivegroups each contain amino acids that are conservative substitutions forone another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine(L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y),Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic:Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D),Glutamic acid (E), Asparagine (N), Glutamine (Q).

A linker or spacer, such as a repeating poly(ethylene glycol) linker orany other suitable linker, may be used to increase distance frompolypeptide to surface of microcarrier. The linker may be of anysuitable length. For example, if the linker is a repeating poly(ethyleneglycol) linker, the linker may contain between 2 and 10 repeatingethylene glycol units. In some embodiments, the linker is a repeatingpoly(ethylene glycol) linker having about 4 repeating ethylene glycolunits. All, some, or none of the polypeptides may be conjugated to amicrocarrier via linkers. Other potential linkers that may be employedinclude polypeptide linkers such as poly(glycine) or poly(β-alanine).

A polypeptide may be conjugated to the microcarrier at any density,preferably at a density suitable to support culture of undifferentiatedstem cells or other cell types. For example, polypeptides may beconjugated to a microcarrier at a density of between about 1 pmol permm² and about 50 pmol per mm² of surface of the microcarrier. Forexample, the polypeptide may be present at a density of greater than 5pmol/mm², greater than 6 pmol/mm², greater than 7 pmol/mm², greater than8 pmol/mm², greater than 9 pmol/mm², greater than 10 pmol/mm², greaterthan 12 pmol/mm², greater than 15 pmol/mm², or greater than 20 pmol/mm²of the surface of the microcarrier. It will be understood that theamount of polypeptide present can vary depending on the composition ofthe microcarrier, the size of the microcarrier, the nature of thepolypeptide itself, and the type of cell to be cultured.

While not intending to be bound by theory, it is believed that stemcells, including embryonic stem cells, may require higher peptidedensities to support cell attachment than some other types of cells,such as, for example, HT1080 cells (see, e.g., EXAMPLE 17). In addition,cells may require higher peptide density to sufficiently attach tomicrocarriers under stirred conditions relative to static conditions. Invarious embodiments, microcarriers have greater than 50 nmol conjugatedpolypeptide per milligram of microcarrier base. For example, amicrocarrier may have greater than 60, greater than 70, greater than 80,greater than 90, or greater than 100 nmol conjugated polypeptide permilligram of microcarrier base. In some embodiments where themicrocarriers are intended to be used for culturing stem cells, themicrocarriers have greater than 100 nmol conjugated polypeptide permilligram of microcarrier base.

A polypeptide may be conjugated to the polymerized microcarrier via anysuitable technique. A polypeptide may be conjugated to a polymerizedmicrocarrier via an amino terminal amino acid, a carboxy terminal aminoacid, or an internal amino acid. One suitable technique involves1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC)/N-hydroxysuccinimide (NHS) chemistry, as generally known in theart. EDC and NHS or N-hydroxysulfosuccinimide (sulfo-NHS) can react withcarboxyl groups of the swellable (meth)acrylate layer to produce aminereactive NHS esters. EDC reacts with a carboxyl group of the swellable(meth)acrylate layer to produce an amine-reactive O-acylisoureaintermediate that is susceptible to hydrolysis. The addition of NHS orsulfo-NHS stabilizes the amine-reactive O-acylisourea intermediate byconverting it to an amine reactive NHS or sulfo-NHS ester, allowing fora two step procedure. Following activation of the swellable(meth)acrylate layer, the polypeptide may then be added and the terminalamine of the polypeptide can react with the amine reactive ester to forma stable amide bond, thus conjugating the polypeptide to the swellable(meth)acrylate layer. When EDC/NHS chemistry is employed to conjugate apolypeptide to the swellable (meth)acrylate layer, the N-terminal aminoacid is preferably an amine containing amino acid such as lysine,ornithine, diaminobutyric acid, or diaminoproprionic acid. Of course,any acceptable nucleophile may be employed, such as hydroxylamines,hydrazines, hydroxyls, and the like.

EDC/NHS chemistry results in a zero length crosslinking of polypeptideto microcarrier. Linkers or spacers, such as poly(ethylene glycol)linkers (e.g., available from Quanta BioDesign, Ltd.) with a terminalamine may be added to the N-terminal amino acid of polypeptide. Whenadding a linker to the N-terminal amino acid, the linker is preferably aN-PG-amido-PEG_(X)-acid where PG is a protecting group such as the Fmocgroup, the BOC group, the CBZ group or any other group amenable topeptide synthesis and X is 2, 4, 6, 8, 12, 24 or any other discrete PEGwhich may be available.

In various embodiments, a 1 μM-10.000 μM polypeptide fluid composition,such as a solution, suspension, or the like, is contacted with activatedmicrocarriers to conjugate the polypeptide. For example the polypeptideconcentration may be between about 100 μM and about 2000 μM, betweenabout 500 μM and about 1500 μM, or about 1000 μM. It will be understoodthat the volume of the polypeptide composition and the concentration maybe varied to achieve a desired density of polypeptide conjugated to themicrocarrier.

The polypeptide may be cyclized or include a cyclic portion. Anysuitable method for forming cyclic polypeptide may be employed. Forexample, an amide linkage may be created by cyclizing the free aminofunctionality on an appropriate amino-acid side chain and a freecarboxyl group of an appropriate amino acid side chain. Also, adi-sulfide linkage may be created between free sulfhydryl groups of sidechains appropriate amino acids in the peptide sequence. Any suitabletechnique may be employed to form cyclic polypeptides (or portionsthereof). By way of example, methods described in, e.g., WO1989005150may be employed to form cyclic polypeptides. Head-to-tail cyclicpolypeptides, where the polypeptides have an amide bond between thecarboxy terminus and the amino terminus may be employed. An alternativeto the disulfide bond would be a diselenide bond using twoselenocysteines or mixed selenide/sulfide bond, e.g., as described inKoide et al, 1993, Chem. Pharm. Bull. 41(3):502-6; Koide et al., 1993,Chem. Pharm. Bull. 41(9):1596-1600; or Besse and Moroder, 1997, Journalof Peptide Science, vol. 3, 442-453.

Polypeptides may be synthesized as known in the art (or alternativelyproduced through molecular biological techniques) or obtained from acommercial vendor, such as American Peptide Company, CEM Corporation, orGenScript Corporation. Linkers may be synthesized as known in the art orobtained from a commercial vendor, such as discrete polyethylene glycol(dPEG) linkers available from Quanta BioDesign, Ltd.

An example of a polypolypeptide that may be conjugated to a microcarrieris a polypeptide that includes KGGNGEPRGDTYRAY (SEQ ID NO:1), which isan RGD-containing sequence from bone sialoprotein with an additional“KGG” sequence added to the N-terminus. The lysine (K) serves as asuitable nucleophile for chemical conjugation, and the two glycine aminoacids (GG) serve as spacers. Cystine (C), or another suitable aminoacid, may alternatively be used for chemical conjugation, depending onthe conjugation method employed. Of course, a conjugation or spacersequence (KGG or CGG, for example) may be present or absent. Additionalexamples of suitable polypeptides for conjugation with microcarriers(with or without conjugation or spacer sequences) are polypeptides thatinclude NGEPRGDTYRAY, (SEQ ID NO:2), GRGDSPK (SEQ ID NO:3) (shortfibronectin) AVTGRGDSPASS (SEQ ID NO:4) (long FN), PQVTRGDVFTMP (SEQ IDNO:5) (vitronectin), RNIAEIIKDI (SEQ ID NO:6) (lamininβ1),KYGRKRLQVQLSIRT (SEQ ID NO:7) (mLMα1 res 2719-2730), NGEPRGDTRAY (SEQ IDNO:8) (BSP-Y), NGEPRGDTYRAY (SEQ ID NO:9) (BSP), KYGAASIKVAVSADR (SEQ IDNO:10) (mLMα1 res 2122-2132), KYGKAFDITYVRLKF (SEQ ID NO:11) (mLMγ1 res139-150), KYGSETTVKYIFRLHE (SEQ ID NO:12) (mLMγ1 res 615-627),KYGTDIRVTLNRLNTF (SEQ ID NO:13) (mLMγ1 res 245-257), TSIKIRGTYSER (SEQID NO:14) (mLMγ1 res 650-261), TWYKIAFQRNRK (SEQ ID NO:15) (mLMα1 res2370-2381), SINNNRWHSIYITRFGNMGS (SEQ ID NO:16) (mLMα1 res 2179-2198),KYGLALERKDHSG (SEQ ID NO:17) (tsp1 RES 87-96), or GQKCIVQTTSWSQCSKS (SEQID NO:18) (Cyr61 res 224-240).

In some embodiments, the peptide comprises KGGK⁴DGEPRGDTYRATD¹⁷ (SEQ IDNO:19), where Lys⁴ and Asp¹⁷ together form an amide bond to cyclize aportion of the polypeptide; KGGL⁴EPRGDTYRD¹³ (SEQ ID NO:20), where Lys⁴and Asp¹³ together form an amide bond to cyclize a portion of thepolypeptide; KGGC⁴NGEPRGDTYRATC¹⁷ (SEQ ID NO:21), where Cys⁴ and Cys¹⁷together form a disulfide bond to cyclize a portion of the polypeptide;KGGC⁴EPRGDTYRC¹³ (SEQ ID NO:22), where Cys⁴ and Cys¹³ together form adisulfide bond to cyclize a portion of the polypeptide, orKGGAVTGDGNSPASS (SEQ ID NO:23).

In embodiments, the polypeptide may be acetylated or amidated or both.While these examples are provided, those of skill in the art willrecognize that any peptide or polypeptide sequence may be conjugated toa microcarrier as described herein.

2. Cell Culture Articles

Microcarriers as described herein may be used in any suitable cellculture system. Typically microcarriers and cell culture media areplaced in a suitable cell culture article and the microcarriers arestirred or mixed in the media. Suitable cell culture articles includebioreactors, such as the WAVE BIOREACTOR® (Invitrogen), single andmulti-well plates, such as 6, 12, 96, 384, and 1536 well plates, jars,petri dishes, flasks, multi-layered flasks, beakers, plates, rollerbottles, tubes, bags, membranes, cups, spinner bottles, perfusionchambers, bioreactors, CellSTACK® culture chambers (CorningIncorporated) and fermenters.

3. Incubating Cells in Culture Media Having Microcarriers ContainingConjugated Polypeptide

A cell culture article housing culture media containing conjugatedpolypeptide as described above may be seeded with cells. The conjugatedpolypeptide employed may be selected based on the type of cell beingcultured. The cells may be of any cell type. For example, the cells maybe connective tissue cells, epithelial cells, endothelial cells,hepatocytes, skeletal or smooth muscle cells, heart muscle cells,intestinal cells, kidney cells, or cells from other organs, stem cells,islet cells, blood vessel cells, lymphocytes, cancer cells, primarycells, cell lines, or the like. The cells may be mammalian cells,preferably human cells, but may also be non-mammalian cells such asbacterial, yeast, or plant cells.

In numerous embodiments, the cells are stem cells which, as generallyunderstood in the art, refer to cells that have the ability tocontinuously divide (self-renewal) and that are capable ofdifferentiating into a diverse range of specialized cells. In someembodiments, the stem cells are multipotent, totipotent, or pluripotentstem cells that may be isolated from an organ or tissue of a subject.Such cells are capable of giving rise to a fully differentiated ormature cell types. A stem cell may be a bone marrow-derived stem cell,autologous or otherwise, a neuronal stem cell, or an embryonic stemcell. A stem cell may be nestin positive. A stem cell may be ahematopoietic stem cell. A stem cell may be a multi-lineage cell derivedfrom epithelial and adipose tissues, umbilical cord blood, liver, brainor other organ. In various embodiments, the stem cells are pluripotentstem cells, such as pluripotent embryonic stem cells isolated from amammal. Suitable mammals may include rodents such as mice or rats,primates including human and non-human primates. In various embodiments,the microcarrier with conjugated polypeptide supports undifferentiatedculture of embryonic stem cells for 5 or more passages, 7 or morepassages, or 10 or more passages. Typically stems cells are passaged toa new surface after they reach about 75% confluency. The time for cellsto reach 75% confluency is dependent on media, seeding density and otherfactors as know to those in the art.

Because human embryonic stem cells (hESC) have the ability to growncontinually in culture in an undifferentiated state, the hESC for usewith microcarriers as described herein may be obtained from anestablished cell line. Examples of human embryonic stem cell lines thathave been established include, but are not limited to, H1, H7, H9, H13or H14 (available from WiCell established by the University ofWisconsin) (Thompson (1998) Science 282:1145); hESBGN-01, hESBGN-02,hESBGN-03 (BresaGen, Inc., Athens, Ga.); HES-1, HES-2, HES-3, HES-4,HES-5, HES-6 (from ES Cell International, Inc., Singapore); HSF-1, HSF-6(from University of California at San Francisco); I 3, I 3.2, I 3.3, I4, I 6, I 6.2, J 3, J 3.2 (derived at the Technion-Israel Institute ofTechnology, Haifa, Israel); UCSF-1 and UCSF-2 (Genbacev et al., Fertil.Steril. 83(5):1517-29, 2005); lines HUES 1-17 (Cowan et al., NEJM350(13):1353-56, 2004); and line ACT-14 (Klimanskaya et al., Lancet,365(9471):1636-41, 2005). Embryonic stem cells may also be obtaineddirectly from primary embryonic tissue. Typically this is done usingfrozen in vitro fertilized eggs at the blastocyst stage, which wouldotherwise be discarded.

Other sources of pluripotent stem cells include induced primatepluripotent stem (iPS) cells. iPS cells refer to cells, obtained from ajuvenile or adult mammal, such as a human, that are geneticallymodified, e.g., by transfection with one or more appropriate vectors,such that they are reprogrammed to attain the phenotype of a pluripotentstem cell such as an hESC. Phenotypic traits attained by thesereprogrammed cells include morphology resembling stem cells isolatedfrom a blastocyst as well as surface antigen expression, gene expressionand telomerase activity resembling blastocyst derived embryonic stemcells. The iPS cells typically have the ability to differentiate into atleast one cell type from each of the primary germ layers: ectoderm,endoderm and mesoderm. The iPS cells, like hESC, also form teratomaswhen injected into immuno-deficient mice, e.g., SCID mice. (Takahashi etal., (2007) Cell 131(5):861; Yu et al., (2007) Science 318:5858).

To maintain stem cells in an undifferentiated state it may be desirableto minimize non-specific interaction or attachment of the cells with thesurface of the microcarrier, while obtaining selective attachment to thepolypeptide(s) attached to the surface. The ability of stem cells toattach to the surface of a microcarrier without conjugated polypeptidemay be tested prior to conjugating polypeptide to determine whether themicrocarrier provides for little to no non-specific interaction orattachment of stem cells. Once a suitable microcarrier has beenselected, cells may be seeded in culture medium containing themicrocarriers.

Prior to seeding, the cells, regardless of cell type, may be harvestedand suspended in a suitable medium, such as a growth medium in which thecells are to be cultured once seeded. For example, the cells may besuspended in and cultured in a serum-containing medium, a conditionedmedium, or a chemically-defined medium. As used herein,“chemically-defined medium” means cell culture media that contains nocomponents of unknown composition. Chemically defined cell culture mediamay, in various embodiments, contain no proteins, hydrosylates, orpeptides of unknown composition. In some embodiments, chemically definedmedia contains polypeptides or proteins of known composition, such asrecombinant growth hormones. Because all components ofchemically-defined media have a known chemical structure, variability inculture conditions and thus variability in cell response can be reduced,increasing reproducibility. In addition, the possibility ofcontamination is reduced. Further, the ability to scale up is madeeasier due, at least in part, to the factors discussed above. Chemicallydefined cell culture media are commercially available from Invitrogen(Invitrogen Corporation, 1600 Faraday Avenue, PO Box 6482, Carlsbad,Calif. 92008) as STEM PRO, a fully serum- and feeder-free (SFM)specially formulated from the growth and expansion of embryonic stemcells, Xvivo (Lonza), and Stem Cell Technologies, Inc. as mTeSR™1maintenance media for human embryonic stem cells.

One or more growth or other factors may be added to the medium in whichcells are incubated with the microcarriers conjugated to polypeptide.The factors may facilitate cellular proliferation, adhesion,self-renewal, differentiation, or the like. Examples of factors that maybe added to or included in the medium include muscle morphogenic factor(MMP), vascular endothelium growth factor (VEGF), interleukins, nervegrowth factor (NGF), erythropoietin, platelet derived growth factor(PDGF), epidermal growth factor (EGF), activin A (ACT) such as activinA, hematopoietic growth factors, retinoic acid (RA), interferons,fibroblastic growth factors, such as basic fibroblast growth factor(bFGF), bone morphogenetic protein (BMP), peptide growth factors,heparin binding growth factor (HBGF), hepatocyte growth factor, tumornecrosis factors, insulin-like growth factors (IGF) I and II,transforming growth factors, such as transforming growth factor-β1(TGFβ1), and colony stimulating factors.

The cells may be seeded at any suitable concentration. Typically, thecells are seeded at about 10,000 cells/cm² of microcarrier to about500,000 cells/cm². For example, cells may be seeded at about 50,000cells/cm² of substrate to about 150,000 cells/cm². However, higher andlower concentrations may readily be used. The incubation time andconditions, such as temperature, CO₂ and O₂ levels, growth medium, andthe like, will depend on the nature of the cells being cultured and canbe readily modified. The amount of time that the cells are cultured withthe microcarriers may vary depending on the cell response desired.

The cultured cells may be used for any suitable purpose, including (i)obtaining sufficient amounts of undifferentiated stem cells cultured ona synthetic surface in a chemically defined medium for use ininvestigational studies or for developing therapeutic uses, (ii) forinvestigational studies of the cells in culture, (iii) for developingtherapeutic uses, (iv) for therapeutic purposes, (v) for studying geneexpression, e.g. by creating cDNA libraries, (vi) for studying drug andtoxicity screening, and (vii) the like.

One suitable way to determine whether cells are undifferentiated is todetermine the presence of the OCT4 marker. In various embodiments, theundifferentiated stems cells cultured on microcarriers as describedherein for 5, 7, or 10 or more passages retain the ability to bedifferentiated.

4. Synopsis

The microcarriers described herein have microcarrier bases formed frommonomers comprising (i) an uncharged hydrophilic unsaturated monomerhaving a hydroxyl group, (ii) a hydrophilic carboxylic acid containingunsaturated monomer, and (iii) a hydrophilic multifunctional unsaturatedmonomer. Any suitable monomers may be used. In various embodiments, themonomers are hydrophilic (meth)acrylate monomers or hydrophilic(meth)acrylamide monomers.

For example, the uncharged hydrophilic unsaturated monomer having ahydroxyl group may be a monomer having Formula I or Formula II asdescribed above. Examples of suitable uncharged hydrophilic unsaturatedmonomers that may be employed include hydroxypropyl methacrylate,2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, glycerolmethacrylate, hydroxypropyl acrylate, 4-hydroxybutyl acrylate,N-(hydroxymethyl)acrylamide, N-[Tris(hydroxymethyl)methyl]acrylamide,3-acryloylamino-1-propanol, N-acrylamido-ethoxyethanol, andN-hydroxyethyl acrylamide.

The hydrophilic carboxylic acid containing unsaturated monomer may bemonomers according to Formula III or IV described above. Specificexamples include 2-carboxyethyl methacrylate, 2-carboxyethyl acrylate,acrylic acid, methacrylic acid, 2-carboxyethyl acrylamide, andacrylamidoglycolic acid.

Examples of suitable hydrophilic multifunctional unsaturated monomerinclude di-, tri- or higher functional (meth)acrylate or(meth)acrylamide monomers such as N,N′methylenebisacrylamide,N,N′(1,2dihydroxyethylene)bisacrylamide, polyethylene glycoldi(meth)acrylate, triglycerol diacrylate, propylene glycol glycerolatediacrylate, trimethylolpropane ethoxylate triacrylate, glycerol1,3-diglycerolate diacrylate. As described above, it may be desirable toinclude a hydrophobic crosslinking monomer, which may be any suitablehydrophobic crosslinking monomer, such as a di-, tri-, or higherfunctional (meth)acrylate or (meth)acrylamide monomers. An example ofhydrophobic cross-linking monomers that may be employed istetra(ethylene glycol) dimethacrylate. In various embodiments, thehydrophilic multifunctional unsaturated monomer is more hydrophilic thantetra(ethylene glycol) dimethacrylate or tetra(ethylene glycol)diacrylate.

The microcarrier base may be formed from any suitable mixture of themonomers discussed above. In various embodiments, the mixture ofmonomers used to form the microcarrier includes (i) about 30 to about 70parts per weight of the uncharged hydrophilic unsaturated monomer havinga hydroxyl group; (ii) about 20 to about 60 parts per weight of thehydrophilic carboxylic acid containing unsaturated monomer; and (iii) 1to 15 parts by weight of the hydrophilic multifunctional unsaturatedmonomer. A hydrophilic multifunctional unsaturated monomer (e.g.,methylene bisacrylamide or dihydroxyethylene bisacrylamide) may be usedin combination with a less hydrophilic or hydrophobic unsaturatedmonomer (e.g., tetra(ethylene glycol) dimethacrylate). In many of suchembodiments, the total crosslinker (multifunctional unsaturated monomer)does not exceed 15 parts by weight of the mixture of monomers, with themore hydrophilic multifunctional unsaturated monomer being at least 1%by weight of the mixture of monomers.

Various embodiments of the microcarrier bases have an EWC of greaterthan about 70%, greater than about 75%, greater than about 80%, greaterthan about 85%, or greater than about 90% in water.

In various embodiments, the pendant carboxyl content (from thehydrophilic carboxylic acid containing unsaturated monomer) of thepolymeric microcarrier is greater than 1, greater than 2, greater than3, or about 4 milliequivalents per gram.

The present disclosure described and contemplates, compositionscomprising the various monomers for forming the microcarriers. Forexample, a composition for forming a microcarrier (whether viawater-in-oil emulsion copolymerization or other method) may include (i)an uncharged hydrophilic unsaturated monomer having a hydroxyl group,(ii) a hydrophilic carboxylic acid containing unsaturated monomer, and(iii) a hydrophilic multifunctional unsaturated monomer, such as thosedescribed above. The monomers may be present in the composition is anysuitable ratio, such as those described above with regard to themicrocarrier bases.

The microcarrier base may be prepared according to any suitable process.In various embodiments, water-in-oil emulsion copolymerization is usedto form the microcarrier base. Using such a reaction scheme and system,hydrophilic monomers can be dissolved in water and added to an oil orhydrophobic solution or suspension. Any suitable hydrophobic liquid,such as octanol, toluene, alkane, such as heptane, hexane or higheralkane including decane, dodecane, hexadecane, heavy mineral oil,silicone oil, fluorinated solvent, or the like may be used. Anemulsifier may be added to promote the formation of the water-in-oilemulsion. Water insoluble (oil soluble) emulsifiers are preferred suchas those having HLB value (hydrophilic lipophilic balance) lower than 9.Examples of such emulsifiers are Sorbitan trioleate (Span 85) HLB=1.8,Sorbitan tristearate (Span 65) HLB=2.1, Sorbitan sesquioleate (Arlacel83) HLB=3.7, Glyceryl monostearate, HLB=3.8, Sorbitan monooleate, (Span80) HLB=4.3, Sorbitan monostearate, (Span 60) HLB=4.7, Sorbitanmonopalmitate, (Span 40) HLB=6.7 Sorbitan monolaurate, (Span 20)HLB=8.6. Hydrophobically modified water soluble polymers or randomcopolymers or block or graft copolymer which are built of components ofdifferent polarity are further examples of suitable emulsifiers. Oneexample of a hydrophobically modified water soluble polymers emulsifieris ethyl cellulose. Of course other such emulsifiers may be employed.

The microcarriers described herein may also include a polypeptideconjugated to the microcarrier base. Any suitable process may beemployed to conjugate the polypeptide. In various embodiments, thepolypeptide is conjugated to a carboxylic acid group resulting from thehydrophilic carboxylic acid containing unsaturated monomer. EDC/NHS orany other suitable chemistry may be used to conjugate the polypeptide toa carboxylic acid group of the microcarrier base. A microcarrier mayhave greater than 60, greater than 70, greater than 80, greater than 90,or greater than 100 nmol conjugated polypeptide per milligram ofmicrocarrier base. In some embodiments where the microcarriers areintended to be used for culturing stein cells, the microcarriers havegreater than 100 nmol conjugated polypeptide per milligram ofmicrocarrier base.

Any suitable polypeptide may be conjugated to the microcarrier base.Preferably, the polypeptide facilitates cell adhesion to themicrocarrier in a bio-specific manner. In various embodiments, thepolypeptide includes an amino acid sequence, or portion thereof,recognized by proteins from the integrin family or leading to aninteraction with cellular molecules able to sustain cell adhesion. Forexample, the polypeptide may include an amino acid sequence derived fromcollagen, keratin, gelatin, fibronectin, vitronectin, laminin, bonesialoprotein (BSP), or the like, or portions thereof. In variousembodiments, polypeptide includes an amino acid sequence of ArgGlyAsp(RGD).

Examples of polypeptides that may be conjugated to a microcarrier baseinclude KGGNGEPRGDTYRAY (SEQ ID NO:1); NGEPRGDTYRAY, (SEQ ID NO:2);GRGDSPK (SEQ ID NO:3); AVTGRGDSPASS (SEQ ID NO:4); PQVTRGDVFTMP (SEQ IDNO:5); RNIAEIIKDI (SEQ ID NO:6); KYGRKRLQVQLSIRT (SEQ ID NO:7);NGEPRGDTRAY (SEQ ID NO:8); NGEPRGDTYRAY (SEQ ID NO:9); KYGAASIKVAVSADR(SEQ ID NO:10); KYGKAFDITYVRLKF (SEQ ID NO:11); KYGSETTVKYIFRLHE (SEQ IDNO:12); KYGTDIRVTLNRLNTF (SEQ ID NO:13); TSIKIRGTYSER (SEQ ID NO:14);TWYKIAFQRNRK (SEQ ID NO:15); SINNNRWHSIYITRFGNMGS (SEQ ID NO:16);KYGLALERKDHSG (SEQ ID NO:17); GQKCIVQTTSWSQCSKS (SEQ ID NO:18);KGGK⁴DGEPRGDTYRATD¹⁷ (SEQ ID NO:19), where Lys⁴ and Asp¹⁷ together forman amide bond to cyclize a portion of the polypeptide; KGGL⁴EPRGDTYRD¹³(SEQ ID NO:20), where Lys⁴ and Asp¹³ together form an amide bond tocyclize a portion of the polypeptide; KGGC⁴NGEPRGDTYRATC¹⁷ (SEQ IDNO:21), where Cys⁴ and Cys¹⁷ together form a disulfide bond to cyclize aportion of the polypeptide; KGGC⁴EPRGDTYRC¹³ (SEQ ID NO:22), where Cys⁴and Cys¹³ together form a disulfide bond to cyclize a portion of thepolypeptide, KGGAVTGDGNSPASS (SEQ ID NO:23); andAc-Lys-Gly-Gly-Pro-Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH₂ (SEQID NO:24).

A polypeptide may be conjugated to the microcarrier at any density,preferably at a density suitable to support culture of undifferentiatedstein cells or other cell types. Polypeptides may be conjugated to amicrocarrier at a density of between about 1 pmol per mm² and about 50pmol per mm² of surface of the microcarrier. For example, thepolypeptide may be present at a density of greater than 5 pmol/mm²,greater than 6 pmol/mm², greater than 7 pmol/mm², greater than 8pmol/mm², greater than 9 pmol/mm², greater than 10 pmol/mm², greaterthan 12 pmol/mm², greater than 15 pmol/mm², or greater than 20 pmol/mm²of the surface of the microcarrier.

The microcarriers described herein may be employed in any suitablesystem. Such as bio-reactors, single and multi-well plates, jars, petridishes, flasks, beakers, roller bottles, tubes, bags, membranes, cups,spinner bottles, perfusion chambers, fermenters, and the like.

The microcarriers, in an appropriate system, may be used to culture anysuitable cells, such as mammalian cells, preferably human cells, ornon-mammalian cells such as bacterial, yeast, or plant cell. Examples ofmammalian cells that may be cultured with the microcarriers describedherein include connective tissue cells, epithelial cells, endothelialcells, hepatocytes, skeletal or smooth muscle cells, heart muscle cells,intestinal cells, kidney cells, or cells from other organs, stem cells,islet cells, blood vessel cells, lymphocytes, cancer cells, primarycells, cell lines, or the like. In numerous embodiments, the cells arestem cells which, as such as multipotent, totipotent, or pluripotentstem cells. In some embodiments the cells are embryonic stem cells. Asshown herein, the described microcarriers may be used to cultureembryonic stem cells in chemically-defined medium.

The cultured cells may be used for any suitable purpose, including (i)obtaining sufficient amounts of undifferentiated stem cells cultured ona synthetic surface in a chemically defined medium for use ininvestigational studies or for developing therapeutic uses, (ii) forinvestigational studies of the cells in culture, (iii) for developingtherapeutic uses, (iv) for therapeutic purposes, (v) for studying geneexpression, e.g. by creating cDNA libraries, (vi) for studying drug andtoxicity screening, and (vii) the like.

In a first aspect, a microcarrier for cell culture, comprising: apolymeric microcarrier base formed from copolymerization of a mixture ofmonomers including (i) an uncharged hydrophilic unsaturated monomerhaving a hydroxyl group selected from a hydrophilic (meth)acrylatemonomer having a hydroxyl group or a hydrophilic (meth)acrylamidemonomer having a hydroxyl group, (ii) a hydrophilic carboxylic acidcontaining unsaturated monomer selected from a carboxylic acidcontaining (meth)acrylate monomer or a carboxylic acid containing(meth)acrylamide monomer, and (iii) a first hydrophilic multifunctionalunsaturated monomer selected from a hydrophilic multifunctional(meth)acrylate monomer or a hydrophilic multifunctional (meth)acrylamidemonomer; and a polypeptide conjugated to the microcarrier base, whereinthe microcarrier base has an equilibrium water content of greater than75% is provided. In a second aspect, a microcarrier according to aspect1, wherein the mixture of monomers includes (i) 30 to 70 parts perweight of the uncharged hydrophilic unsaturated monomer having ahydroxyl group; (ii) 20 to 60 parts per weight of the hydrophiliccarboxylic acid containing unsaturated monomer; and (iii) 1 to 15 partsby weight of the first hydrophilic multifunctional unsaturated monomeris provided. In a third aspect, a microcarrier according to aspect 1 or2, wherein the uncharged hydrophilic unsaturated monomer having ahydroxyl group is a monomer according to Formula (I) or Formula (II):

where A is H or methyl, and where B is C1-C6 straight or branched chainalcohol or ether. In some embodiments, B is C1-C4 straight or branchedchain alcohol; or

where A is hydrogen or methyl, and where B is C1-C6 straight or branchedchain alcohol or ether is provided. In a fourth aspect, a microcarrieraccording to aspect 1 or 2, wherein the uncharged hydrophilicunsaturated monomer having a hydroxyl group is selected from the groupconsisting of hydroxypropyl methacrylate, 2-hydroxyethyl methacrylate,2-hydroxyethyl acrylate, glycerol methacrylate, hydroxypropyl acrylate,4-hydroxybutyl acrylate, N-(hydroxymethyl)acrylamide,N-[Tris(hydroxymethyl)methyl]acrylamide, 3-acryloylamino-1-propanol,N-acrylamido-ethoxyethanol, and N-hydroxyethyl acrylamide is provided.In a fifth aspect, a microcarrier according to aspect 1 or 2, whereinthe uncharged hydrophilic unsaturated monomer having a hydroxyl group is2-hydroxyethyl methacrylate is provided. In a sixth aspect, amicrocarrier according to any of aspects 1-5, wherein the hydrophiliccarboxylic acid containing unsaturated monomer is a monomer according toFormula (III) or Formula (IV):

where A is hydrogen or methyl, and where D is C1-C6 straight or branchedchain alkyl substituted with a carboxyl group (—COOH); or

where A is hydrogen or methyl, and where D is C1-C6 straight or branchedchain alkyl substituted with a carboxyl group (—COOH). In a seventhaspect, microcarrier according to any of aspects 1-5, wherein thehydrophilic carboxylic acid containing unsaturated monomer is selectedfrom the group consisting of 2-carboxyethyl methacrylate, 2-carboxyethylacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylamide, andacrylamidoglycolic acid is provided. In an eighth aspect, a microcarrieraccording to any of aspects 1-5, wherein the hydrophilic carboxylic acidcontaining unsaturated monomer is 2-carboxyethyl methacrylate. In aninth aspect, microcarrier according to any of aspects 1-8, wherein thefirst hydrophilic multifunctional unsaturated monomer is selected fromthe group consisting of N,N′methylenebisacrylamide,N,N′(1,2-dihydroxyethylene)bisacrylamide, polyethylene glycoldi(meth)acrylate, triglycerol diacrylate, propylene glycol glycerolatediacrylate, trimethylolpropane ethoxylate triacrylate, and glycerol1,3-diglycerolate diacrylate is provided. In a tenth aspect, amicrocarrier according to any of aspects 1-8, wherein the firsthydrophilic multifunctional unsaturated monomer is selected from thegroup consisting of methylene bisacrylamide and dihydroxyethylenebisacrylamide is provided. In an eleventh aspect a microcarrieraccording to any of aspects 1-10, wherein the mixture of monomersfurther comprises a second multifunctional unsaturated monomer selectedfrom a multifunctional unsaturated (meth)acrylate monomer or amultifunctional unsaturated (meth)acrylamide monomer is provided. In atwelfth aspect, a microcarrier according to aspect 11, wherein thesecond multifunctional unsaturated monomer is less hydrophilic than thefirst hydrophilic multifunctional unsaturated monomer is provided. In athirteenth aspect, a microcarrier according to aspect 11 or claim 12,wherein the second multifunctional unsaturated monomer is tetra(ethyleneglycol) dimethacrylate is provided. In a fourteenth aspect, amicrocarrier according to any of aspects 11-13, wherein the firsthydrophilic multifunctional unsaturated monomer and the secondmultifunctional unsaturated monomer together do not exceed 15 parts byweight of the mixture of monomers. In a fifteenth aspect, a microcarrieraccording to any of aspects 1-14, wherein the polypeptide is anRGD-containing polypeptide is provided. In a sixteenth aspect, amicrocarrier according to any of aspects 1-14, wherein the polypeptideis selected from the group consisting of a BSP polypeptide, avitronectin polypeptide, a fibronectin polypeptide, and a collagenpolypeptide is provided. In a seventeenth aspect, a microcarrieraccording to any of aspects 1-16, wherein the microcarrier has greaterthan 100 nmol conjugated polypeptide per milligram of microcarrier baseis provided. In an eighteenth aspect, a microcarrier of any of aspects1-17, wherein the microcarrier base is monolithic is provided. In anineteenth aspect, a method for culturing human embryonic stem cellscomprising: contacting the cells with a cell culture medium havingmicrocarriers according to any of claims 1-18; and culturing the cellsin the medium is provided. In a twentieth aspect, a method according toaspect 19, wherein the microcarrier base is formed from a mixture ofmonomers comprising is 2-hydroxyethyl methacrylate, 2-carboxyethylmethacrylate, and (i) methylene bisacrylamide or (ii) dihydroxyethylenebisacrylamide, wherein the mixture of monomers includes (i) 30 to 70parts per weight of the 2-hydroxyethyl methacrylate; (ii) 20 to 60 partsper weight of the 2-carboxyethyl methacrylate; and (iii) 1 to 15 partsby weight of methylene bisacrylamide or dihydroxyethylene bisacrylamide,and wherein the polypeptide is an RGD-containing polypeptide isprovided. In a twenty-first aspect, a method according to aspect 20,wherein the polypeptide is a vitronectin polypeptide is provided. In atwenty-second aspect, a method according to aspect 20 or 21, wherein themixture of monomers further comprises tetra(ethyleneglycol)dimethacrylate, and wherein the combination of the tetra(ethyleneglycol)dimethacrylate and the methylene bisacrylamide or thetetra(ethylene glycol)dimethacrylate and the dihydroxyethylenebisacrylamide does not exceed 15 parts by weight of the mixture ofmonomers is provided.

In the following, non-limiting examples are presented, which describevarious embodiments of the microcarriers and methods discussed above.

EXAMPLES Example 1 Microcarrier Preparation

250 grams of dry toluene and 15 grams of SPAN 85 emulsifier were chargedin a 500 milliliter reactor equipped with a thermostatic jacket andbottom drain, dropping funnel, anchor stirrer and inert gas bubblingtube. The temperature of the reactor was set at 40° C. and the mixturewas stirred at 500 rpm under argon bubbling for at least 15 min. Then ahomogenous mixture containing 22 milliliter of deionized water, 6.3grams of 2-hydroxyethyl methacrylate, 1.8 grams of 2-carboxyethylacrylate sodium salt and 1 gram methylene bisacrylamide adjusted at pH8-10 with NaOH was prepared. 0.5 grams of ammonium persulfate wasdissolved in this aqueous solution until a clear and homogeneoussolution was obtained. Then this solution was added dropwise by means ofthe dropping funnel to the stirred toluene/SPAN85 mixture at 40° C. Themixture turned milky rapidly due to the water-in-oil emulsion formation.After 15 min mixing, 100 microliters of tetramethylethylene diamine wasadded quickly to the stirred emulsion. After 2 hours of reaction, thetemperature was cooled down to 20° C. and the microcarriers werewithdrawn from the reactor by the bottom drain of the reactor. Themicrocarriers obtained were thoroughly washed with acetone in order toremove the water insoluble emulsifier and unreacted materials. Afterdrying the microcarrier powder was a white, free flowing powder thatcould be stored at RT without using special condition.

The microcarriers, without sieving, were viewed under a microscope andappeared to be generally spherical with a narrow size distribution. Arepresentative image is shown in FIG. 1.

The microcarriers were then viewed with the aid of a scanning electronmicroscope.

Briefly, the beads were metalized with 6 nm gold-palladium. Observationswere done using Field effect gun scanning electron microscope (ref.LEO1550). The surface of the spherical microcarriers appeared smooth andnon-porous (see FIGS. 2A and B showing 500× (FIG. 2A) and 2,000× (FIG.2B) magnification images, respectively. FIG. 3 shows SEM images of themicrospheres obtained after PBS buffer washing at differentmagnifications.

The microcarriers were then subjected to laser diffraction particle sizeanalyzer for determination of size distribution. The analysis wasperformed using a multi-wavelength Beckman Coulter™ LS 13 320 equippedwith an aqueous liquid module and tap water. A graph of the sizedistribution of the particles is shown in FIG. 4. The particles obtainedhad a diameter of 101.8 micrometers (+/−24.42 micrometers). Examples 6,8, and 9 (see below) discuss synthesis of microcarriers having a largerdiameters

The microspheres were also swelled in water (1.5 cubic centimeters ofdry microcarrier in 20 milliliters of water). The microspheres swelledto a volume of about 4.5 cubic centimeters.

Example 2 Assessment of COOH Content Using Crystal Violet

10 micrograms of dry microcarriers prepared according to Example 1 weresuspended in 2 milliliters of deionized water and 10 microliters of 5%wt/v crystal violet aqueous solution was added. After sedimentation, thestained microcarriers were washed with deionized water until a colorlesssupernatant was obtained. The washed microcarriers were suspended in 2milliliters deionized water. Uniform distribution of staining wasobserved (data not shown), suggesting that carboxyl groups were evenlydistributed along the surface of the microcarriers.

Example 3 Amine Coupling Assessed with Alexa Fluor™ 488 Coupling

10 micrograms of microcarriers prepared according to Example 1 wereweighed in an Eppendorf plastic tube and 900 microliters of water wasadded to disperse the microcarriers by shaking. Then 100 microliters of200 mM EDC and 50 mM NHS or Sulfo-NHS at was added. The activation wasperformed for 30 min. The microcarriers were rinsed with 1 ml deionizedwater after microcarrier sedimentation by centrifugation. Rinsing wasrepeated three times. Then the activated microcarriers were suspended in400 microliters borate buffer pH 9.2 and 100 microliters of 1 mM AlexaFluor™ 488 from Invitrogen Molecular Probes™ was added. After one hourcoupling the microcarriers were collected by centrifugation and rinsedthree times with PBS buffer.

Substantial fluorescence was observed with EDC/NHS and sulfo-EDC/NHSamine mediated coupling (data not shown), suggesting that either can beused to conjugate polypeptides to the surface of the microspheres.Sulfo-EDC/NHS resulted in slightly more fluorescence than EDC/NHS.

Example 4 Peptide Grafting

For purposes of proof-of-concept, GRGDS(SEQ ID NO:26) peptide wasgrafted on microcarriers prepared as described in Example 1, usingEDC/Sulfo NHS or EDC/NHS mediated coupling. As described above in thespecification, other peptides of interest, such as those containingamino acid sequences potentially recognized by proteins from theintegrin family, or leading to an interaction with cellular moleculesable to sustain cell adhesion, can be grafted using a similar protocol.Briefly, 10 micrograms of dry microcarriers were weighed in an Eppendorftube and dispersed in 900 microliters of deionized water. Then 100microliters of 20 mM EDC and 5 mM sulfo-NHS were added and letundisturbed for 30 min. to activate the carboxylic acid groups. Theactivated microcarriers were collected by centrifugation and rinsedthree times with deionized water. They were then resuspended in 400microliters borate buffer (pH 9) and 100 microliters 5 mM GRGDS (SEQ IDNO:26) peptide was added and left to react for 1 hour.

The microcarriers were collected by centrifugation and washed threetimes with 1 ml PBS buffer pH 7.4. Finally, the excess activated esterwas deactivated by blocking with 1 ml ethanolamine for 30 minutes. Thepeptide grafted and blocked microcarriers were collected and rinsedthree times with PBS. After rinsing with PBS the microcarriers wererinsed 2 times with 70:30% v/v ethanol/water and stored in thisethanol/water solution before cell culture.

Example 5 Cell Culture on Peptide Grafted Microcarriers

CHO-M1 cells (ATCC #CRL-1984) were grown in F-12 Kaighn's modifiedmedium (Gibco) supplemented with 10% fetal bovine serum (Hyclone) andpenicillin/streptomycin. Cells were kept in an incubator at 37° C. with5% CO2 in a humid atmosphere, medium was changed every 3 days, and cellswere trypsinized and diluted when needed to remain below confluency.

Before cell seeding, microcarriers prepared according to Example 4 keptin an ethanol/water solution (70/30) were washed 3 times in HBSS buffer(Gibco). Finally, the microcarriers were suspended in cell culturemedium and dispensed in a Corning® ULA plate.

Cells for seeding on microcarriers were collected during theirexponential growth phase by tryspination. The cells were counted andwashed with HBSS buffer. The cell concentration was adjusted to 10⁶cells/milliliter, and the cells were dispensed on the microcarriers andincubated for at least 4 hours in an incubator.

Cell adhesion on the peptide-grafted microcarriers were observed afterthe initial 4 hours incubation. Images of CHO-M1 adhering on thecarriers are presented in FIG. 5. Image A presents the cells 4 hoursafter seeding: the rounded shape of the cells is characteristic of theearly steps of adhesion. After 16 hours of incubation (image B) the cellspreading on the surface is evident suggesting integrin engagement andfocal adhesion formation. Cells at this stage should be transferred tosuspension culture vessels for further growth.

HEK293 (ATCC #crl-1573) cells were seeded on microcarriers at differentstages of peptides grafting and cultured in DMEM Gibco+10% FBS(Hyclone). Cell adhesion was not observed in the absence of peptide andethanolamine treatment (not shown). FIG. 6 is an image of HEK293 cellsadhering on microcarriers grafted with GRGDS peptide. Briefly HEK293cells were seeded on microcarriers at different stages of peptidesgrafting. Cell adhesion was not observed in the absence of peptide andethanolamine treatment.

MRC5 cells (ATCC #CCL-171) were seeded on microcarriers at differentstages of peptides grafting and were cultured in DMEM Gibco+10% FBS(Hyclone). Cell adhesion was not observed in the absence of peptide andethanolamine treatment (not shown). FIG. 7 is an image of MRC5 cellsadhering on microcarriers grafted with GRGDS (SEQ ID NO:26) peptide.

These results demonstrate the lack of toxicity of the microcarriers usedin these culture conditions.

Example 6 Microcarrier Preparation

250 grams of dry toluene and 15 grams of SPAN 85 emulsifier were chargedin a 500 milliliter reactor equipped with a thermostatic jacket andbottom drain, dropping funnel, anchor stirrer and inert gas bubblingtube. The temperature of the reactor was set at 40° C. and the mixturewas stirred at 260 rpm under argon bubbling for at least 15 min. Then ahomogenous mixture containing 22 milliliter of deionized water, 6.3grams of 2-hydroxyethyl methacrylate, 1.8 grams of 2-carboxyethylacrylate sodium salt and 0.5 gram dihydroxyethylene bisacrylamideadjusted at pH 8-10 with NaOH was prepared. 0.5 grams of ammoniumpersulfate was dissolved in this aqueous solution until a clear andhomogeneous solution was obtained. Then this solution was added dropwiseby means of the dropping funnel to the stirred toluene/SPAN85 mixture at40° C. The mixture turned milky rapidly due to the water-in-oil emulsionformation. After 15 min mixing, 100 microliters of tetramethylethylenediamine was added quickly to the stirred emulsion. After 2 hours ofreaction, the temperature was cooled down to 20° C. and themicrocarriers were withdrawn from the reactor by the bottom drain of thereactor. The microcarriers obtained were thoroughly washed with acetonein order to remove the water insoluble emulsifier and unreactedmaterials. After drying the microcarrier powder was a white powder thatcould be stored at 4° C. to prevent agglomeration of the microspheres.

The microcarriers, without sieving, were viewed under a microscope andappeared to be highly spherical with an acceptable size distribution andvery high transparency. A representative image is shown in FIG. 8.

Example 7 Vitronectin-Peptide Grafting

Vitronectin-peptide was grafted on microcarriers prepared as describedin Example 6, using EDC/Sulfo NHS mediated coupling. Briefly, 10micrograms of dry microcarriers were weighed in an Eppendorf tube anddispersed in 900 microliters of deionized water. Then 100 microliters of200 mM EDC and 50 mM sulfo-NHS were added and let undisturbed for 30minutes to activate the carboxylic acid groups. The activatedmicrocarriers were collected by centrifugation and rinsed three timeswith deionized water. They were then resuspended in 10 mM vitronectinpeptide (ref 341587 (ID #VN) from American peptide Company Inc. CA USA,having an amino acid sequence ofAc-Lys-Gly-Gly-Pro-Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH₂ (SEQID NO:24)) in 1000 microliters borate buffer pH 9.2 was added and leftto react for 2 hours. The microcarriers were collected by centrifugationand washed three times with 1 ml PBS buffer pH 7.4. Finally, the excessactivated ester was deactivated by blocking with 1 ml ethanolamine for30 minutes. The peptide grafted and blocked microcarriers were collectedand rinsed three times with PBS. After rinsing with PBS themicrocarriers were rinsed 2 times with 70:30% v/v ethanol/water andstored in this ethanol/water solution before cell culture.

Example 8 RGE-Peptide Grafting

The same procedure as described in Example 7 was reproduced except thatRGE-peptide (ref 348454 (ID #RGE) from American peptide Company Inc. CAUSA, having amino acid sequenceAc-Gly-Arg-Gly-Glu-Ser-Pro-Ile-Ile-Lys-NH₂ (SEQ ID NO:25)) was utilizedinstead of the vitronectin peptide from Example 7. These microcarriersgrafted with a peptide containing a RGE core were used as a negativecontrol.

Example 9 HT1080 Cell Culture on Vitronectin-Peptide and RGE-PeptideGrafted Microcarriers

HT-1080 cells (ATCC #CCL-121) were grown in IMDM medium (Gibco) with 10%fetal bovine serum (Hyclone).

For adhesion assays, 10 mg of peptide-grafted microcarriers from example7 and 8 were washed in D-PBS, resuspended in serum free culture mediumand transferred to 24 wells multiwell plates. HT1080 cells weretrypsinized, counted and 500,000 cells were mixed with the beadspreviously prepared. Plates were incubated 1 hour at 37° C. and celladhesion was observed using phase contrast microscopy as shown in FIG.9. FIG. 9A shows cells growing on microcarriers having a VN sequence.FIG. 9B shows cell culture with microcarriers with an RGE peptidesequence. Comparison of image A and B reveals that cell adhesion isspecific to the RGD core of the vitronectin peptide.

Example 10 Microcarrier Preparation

The same procedure as in Example 1 was reproduced except that stirringspeed was 260 rpm. An average particle size of 330 μm was observed (seeFIG. 10).

Example 11 Microcarrier Preparation

The same procedure as in Example 1 was reproduced except that stirringspeed was 300 rpm. An average particle size of 210 μm was observed (seeFIG. 10).

Example 12 Microcarrier Preparation (μHG07)

200 grams of dry toluene and 15 grams of SPAN 85 emulsifier were chargedin a 500 milliliter reactor equipped with a thermostatic jacket andbottom drain, dropping funnel, anchor stirrer and inert gas bubblingtube. The temperature of the reactor was set at 40° C. and the mixturewas stirred at 260 rpm under argon bubbling for at least 15 min. Then ahomogenous mixture containing 10 milliliter of deionized water, 3.1grams of 2-hydroxyethyl methacrylate, 5.0 grams of 2-carboxyethylacrylate, 0.15 gram methylene bisacrylamide, 0.40 gramtetraethyleneglycol dimethacrylate and 1 ml ethanol was prepared. The pHwas adjusted at pH 8-9 by adding dropwise about 4.45 gram 10M sodiumhydroxide. Then, 0.63 grams of ammonium persulfate was dissolved in thisaqueous solution until a clear and homogeneous solution was obtained.Then this solution was bubbled for 1 minute with dry argon and addeddrop wise by means of the dropping funnel to the stirred toluene/SPAN85mixture at 40° C. After 15 min mixing, 100 microliters oftetramethylethylene diamine was added quickly to the stirred emulsion.After 4 hours of reaction, the temperature was cooled down to 20° C. andthe microcarriers were withdrawn from the reactor by the bottom drain ofthe reactor. The microcarriers obtained were thoroughly washed withacetone in order to remove the surfactant and unreacted materials. Thebeads were swelled in water for 2 hours and wet-sieved using 100, 200and 400 μM nylon mesh sieve. After air drying the sieved microcarrierhaving size between 200 and 400 μm was used for further peptideconjugation as described below.

Example 13 Vitronectin-Peptide Grafting

Vitronectin-peptide was conjugated to microcarriers prepared accordingto Example 12 using EDC/NHS mediated coupling. Briefly, 10 micrograms ofdry microcarriers from Example 12 were weighed in an Eppendorf tube anddispersed in 900 microliters of deionized water. Then 100 microliters of200 mM EDC and 50 mM NHS were added and let undisturbed for 30 minutesto activate the carboxylic acid groups. The activated microcarriers werecollected by centrifugation and rinsed three times with deionized water.Then 1000 microliters of 2.5 mM vitronectin peptide (ref 341587 (ID #VN)from American peptide Company Inc. CA USA, having an amino acid sequenceof Ac-Lys-Gly-Gly-Pro-Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH₂(SEQ ID NO:24)) in borate buffer pH 9.2 was added and the suspensionleft to react for 30 min, the tube being shaked every 10 minutes. Themicrocarriers were collected by centrifugation and washed three timeswith 1 ml PBS buffer pH 7.4. Finally, the excess activated ester wasdeactivated by blocking with 1 ml 1 M ethanolamine pH 8.4 for 30minutes. The peptide grafted and blocked microcarriers were collectedand rinsed three times with PBS. After rinsing with PBS themicrocarriers were rinsed 2 times with 70:30% v/v ethanol/water andstored in this ethanol/water solution before cell culture. Thesemicrocarriers are referred herein as μHG07.

Example 14 Microcarrier Preparation (μHG14)

200 grams of dry toluene and 15 grams of SPAN 85 emulsifier were chargedin a 500 milliliter reactor equipped with a thermostatic jacket andbottom drain, dropping funnel, anchor stirrer and inert gas bubblingtube. The temperature of the reactor was set at 40° C. and the mixturewas stirred at 330 rpm under argon bubbling for at least 15 min. Then ahomogenous mixture containing 10 milliliter of deionized water, 3.1grams of 2-hydroxyethyl methacrylate, 5.0 grams of 2-carboxyethylacrylate, 0.8 grams of glycerol 1,3-diglycerolate diacrylate wasprepared. The pH was adjusted at pH 8-9 by adding dropwise about 4.46gram 10M sodium hydroxide. Then, 0.63 grams of ammonium persulfate wasdissolved in this aqueous solution until a clear and homogeneoussolution was obtained. Then this solution was bubbled for 1 minute withdry argon and added drop wise by means of the dropping funnel to thestirred toluene/SPAN85 mixture at 40° C. After 15 min mixing, 150microliters of tetramethylethylene diamine was added quickly to thestirred emulsion. After 3 hours of reaction, the temperature was cooleddown to 20° C. and the microcarriers were withdrawn from the reactor bythe bottom drain of the reactor. The microcarriers obtained werethoroughly washed with acetone in order to remove the surfactant andunreacted materials. The beads were swelled in water for 2 hours andwet-sieved using 100, 200 and 400 μm nylon mesh sieve. After air dryingthe sieved microcarrier having size between 200 and 400 μm was used forpeptide conjugation accordingly to the protocol from example 13.

Example 15 Cell Adhesion Assays Using HT1080 Cells

For adhesion assays, 5 mg of VN peptide-grafted microcarriers fromExample 13 were washed in D-PBS, saturated with bovine serum albumin(BSA), resuspended in serum free culture medium and transferred to 24wells multiwell ultralow attachment (ULA) plates.

HT1080 human fibrosarcoma cells (ATCC #CCL-121) were grown in Iscove'sModified Dulbecco's Medium (IMDM) (Gibco) supplemented with 10% fetalbovine serum (FBS) and penicillin streptomycin antibiotics. Cells werekept in an incubator at 37° C., in a humid atmosphere with 5% CO₂. Cellswere split as needed by trypsinization before they reached confluency.

HT1080, cells were trypsinized washed and counted, and 500,000 cells inserum free medium were mixed with the beads previously prepared. Plateswere incubated 2 hours at 37° C. and cell adhesion was observed usingphase contrast microscopy. When quantification was needed, thepreparation was fixed using 3.7% formaldehyde in PBS and images of thecells.

A representative image of HT1080 cells adhered to μHG07 microcarriers isdepicted in FIG. 11, which is a phase contrast microscopy image takentwo hours after seeding. As briefly discussed above with regard toEXAMPLE 9, no adhesion of HT1080 cells was observed on microcarrierswith grafted RGE polypeptide (1HG0X RGE), but was observed on VN peptidegrafted microcarriers, indicating that the cell binding ispeptide-specific and that little to no non-specific binding to themicrocarrier base occurs (see FIG. 9).

The ability of the anchorage-dependent HT1080 cells to bind and expandon μHG0X-VN microcarriers series (i.e., those prepared in the Examplespresented herein) and to remain bound and grow on microcarriers understirred culture conditions was examined and compared to Cytodex™ 1microcarriers (GE Healthcare Biosciences AB Ltd), which are positivelycharged and are believe to support non-specific binding ofanchorage-dependent cells or other commercially available carriersincluding cytodex3 and solohill pronectin F carriers. Briefly, 100 mg ofpeptide-grafted microcarriers, or commercially available microcarriers(cytodex 1, cytodex 3, SoloHill Pronectin® F) were washed in D-PBS,resuspended in culture medium and transferred to a 125 mL spinner flask(Corning, Inc.). HT1080 cells were trypsinized and counted, and2.500,000 cells were mixed with the beads previously prepared. After aninitial 2 hour adhesion period in a final medium volume of 10 mL withoutagitation, the medium volume was adjusted to 30 mL and the agitation wassetup to 15 minutes every hour. Samples were collected daily andevaluated by phase contrast microscopy. As shown in FIG. 12, HT1080cells adhered to and expanded on μHG0X-VN microcarriers as well as, orbetter than, on Cytodex™ 3 beads. μHG07-VN in particular allowed thehighest expansion rate. The phase contrast images in FIG. 12 were taken2 hours, and 4 days after seeding the cells on the μHG07-VNmicrocarriers.

After 4 to 5 days, cell-bearing carriers were collected bycentrifugation, washed in PBS buffer, and the cells were harvested bytrypsinization. The number of living cells was determined by countingwith a Malassez cell after trypan blue exclusion test. The results arepresented in FIG. 13, where the final cell number is reported to thegrowth measured on ProF microcarriers. As shown in FIG. 13, theanchorage dependent HT1080 cells grew better on the μHG07 microcarriersthan on the Cytodex 1™, Cytodex 3™, and ProF microcarriers.

The results presented herein show that hydrogel microcarriers cansupport attachment and growth of anchorage-dependent cells, such asHT1080 cells, in a peptide-specific manner in serum free conditions. Thebinding of the cells to such microcarriers is sufficient to supportculture under stirred conditions. Further, the cells appear to attachand grow better on such microcarriers than on other commerciallyavailable microcarriers.

Example 16 Adhesion of Pluripotent Mouse Embryonic Stem Cells

For long term adhesion assays, 5 mg of VN peptide-grafted microcarriers,microcarriers were washed in D-PBS, resuspended in serum free culturemedium (mTeSR, StemCell Technologies) and transferred to 24 wellsmultiwell ULA plates.

ES-D3 pluripotent mouse stem embryonic stem cells (ATCC #CRL-11632) weregrown in DMEM medium supplemented with 15% FBS and 0.1 mMbeta-mercaptoethanol as recommended by ATCC. Cells were routinely grownon Matrigel coated plates, and trypsinized and diluted before they reachconfluency.

ES-D3 cells were trypsinized, washed and counted. 1×10⁶ cells wereresuspended in mTeSR serum free medium, mixed with the beads previouslyprepared as described in example 12. Plates were incubated for 48 hoursat 37° C., and cell adhesion was observed at 48 hours using phasecontrast microscopy (see FIG. 14). Pluripotent mouse embryonic stemcells are able to adhere nicely to the microcarriers tested. We observethat, despite the lack of agitation, the tendency of the microcarriersto form aggregates is limited.

Cell pluripotency was investigated at the end of the experiment bydosing the activity of alkaline phosphatase, an enzyme specificallyexpressed by pluripotent embryonic stem cells. The results, which arepresented in FIG. 15, indicate that the cells pluripotency is maintainedas expected in a way comparable to what is observed on the positivecontrol (Cytodex3™). STO mouse fibroblasts (ATCC #CRL-1503) grown on TCTas recommended by ATCC are used as a negative control, as expected cellsare not pluripotent and do not express alkaline phosphatase.

ES-D3 mESC were also able to adhere on the microcarriers prepared as inExample 14 as illustrated in FIG. 15.

Those results support the suggestion that the microcarriers describedherein will support culture of pluripotent stem cells, such as humanembryonic stem cells.

Example 16 Adhesion and Expansion of Human Embryonic Stem Cells

BG01V/hOG cells (Invitrogen) were maintained on Matrigel coated TCT 75Flask (Corning) in serum free mTeSR1 medium containing 50 μg/mlHygromycin B (STEMCELL Technologie). Daily medium changes began afterthe first 48 h in culture. Cells were passaged every 5 to 6 days usingcollagenase IV (Invitrogen) and mechanical scraping.

For the assay, aggregate colonies were harvested and resuspended infresh mTeSR1 serum free medium. Cells were seeded to the 24 wellsCorning Ultra low attachment microplate (1.5×105 cells per cm2)containing the VN-peptide grafted μHG07 microcarriers, prepared asdescribed above, or Cytodex™ 3 microcarrier available from GE Healthcareas a comparative example. The volume was adjusted to 600 microliterswith culture medium. Cells were allowed to attach to the microcarriersfor 48 h without agitation. Two days after seeding, cellular attachmentand spreading was assessed using Ziess Axiovert 200M invertedmicroscope.

As shown in FIG. 17, one can observe a nice spreading of BG01V/hOG cellcolonies on μHG07-VN beads. On Cytodex 3 beads the cells were not ableto adhere and instead formed floating embryonic bodies.

For stirred culture, 5×10⁶ cells were seeded as described previously on50 mg of microcarriers in a spinner flask in a final volume of 15 mLmTeSR1 medium, cells were incubated for 2 days without agitation, andthen cultures were stirred gently 15 minutes every 2 hours. 80% of themedium was changed daily beginning after 48 hours. At day 5microcarriers were collected cells were recovered by trypsination andcounted using the trypan blue exclusion method. The results obtained arepresented in FIG. 18, which show that the cell growth observed onhydrogel microcarriers is comparable to what is observed on Matrigelcoated glass beads, used as a gold standard. No significant growth isobserved on non coated glass beads used as a negative control. Thisexperiment demonstrates the suitability of microcarriers as describedherein to support the growth of undifferentiated human embryonic stemcells in stirred conditions for several days.

Example 17 Comparison of Various Microcarriers to Support Cell Adhesion

Microcarriers were formed from a variety of monomers at differingconcentrations and the physical properties and ability various cells toattach to the microcarriers were examined. The microcarrier bases wereprepared as generally described in the previous Examples and vitronectinpolypeptide was conjugated to the microcarrier base to form themicrocarrier for cell culture as described in prior Examples. Theswelling factor and equilibrium water content (EWC) of the microcarrierbase. The peptide density of the resulting microcarrier was evaluated asdescribed in the previous Examples. The ability of HT1080 cells, ESD3cells, and BG01 V/Hog human embryonic stem cells to attach to themicrocarriers under cell culture conditions as described in the previousExamples were also evaluated by microscopic observation.

The monomers employed to form the carious microcarrier bases are listedin Table 1.

TABLE 1 Monomers used to form microcarrier bases μHG07 μHG12 μHG10 μHG11μHG14 HEMA 35.84 34.6 33.37 30.44 34.83 CEA 57.8 55.8 53.82 54.35 56.18MBA 1.74 2.46 3.23 6.52 0 TEGDMA 4.62 7.14 9.58 8.69 0 GDGDA 0 0 0 08.99

The numbers presented in Table 1 represent the amount of monomeremployed in grams; HEMA=2-hydrodyethyl methacrylate; CEA=2-carboxyethylacrylate; MBA=methylene bisacrylamide; TEGDMA=tetra(ethyleneglycol)dimethacrylate; GDGDA=glycerol 1,3-diglycerolate diacrylate.

All the microcarrier bases had about 4 milliequivalents of carboxylicacid functional group per gram, yet the polypeptide density varied amongthe microcarriers even though the processes for conjugating thepolypeptide were the same. The amount of crosslinker used correlatedwith the polypeptide density (see, FIG. 19), with the polypeptidedensity decreasing with increasing crosslinker %. The specific estimatedCOOH functionality, weight percent crosslinker and polypeptide densityfor each microcarrier base is presented in Table 2.

TABLE 2 COOH functionality, crosslinker, and peptide density ofmicrocarriers or microcarrier bases μHG07 μHG12 μHG10 μHG11 μHG14Estimated 4 3.9 3.74 3.78 3.9 COOH (meq/gm) % wt x-linker 6.3 8.2 13 158.99 Peptide loading 153 118 108 59 99 (nmol/mg)

The ability of the microcarriers to swell and the equilibrium watercontent of each of the microcarriers was evaluated and the results arepresented in Table 3.

TABLE 3 Swelling factor and EWC of microcarrier bases μHG07 μHG12 μHG10μHG11 μHG14 Swelling 30 15 10 7 16 factor (ml/gm) EWC (%) 97 93 90 86 94

The swelling factor and EWC of the resulting microcarriers isconsiderably higher than the swellable (meth)acrylate layers formed inthe examples of copending U.S. patent application Ser. Nos. 12/362,924and 12/362,974. This is believed to be due to the increased weightpercent of the carboxylic acid containing monomer used in themicrocarriers described in the present Examples. In copending U.S.patent application Ser. Nos. 12/362,924 and 12/362,974, the swellable(meth)acrylate layers were formed in situ on a substrate. If the EWCwere too high, the layer may swell too much and delaminate. However, asthe microcarriers described herein are not formed as a layer on asubstrate, delamination is not a concern and the EWC can be considerablyhigher. In many respects, microcarriers with higher EWCs are desirablebecause they tend to have low stiffness and thus more closely resemplebiological tissue stiffness, high transparency due to the high amount ofwater, and prevent or reduce cell damage during collision in stirredcultures.

The ability of these microcarriers to support attachment of cellsvaried, as shown in Table 4.

TABLE 4 Ability of cells to attach to microcarriers μHG07 μHG12 μHG10μHG11 μHG14 HT1080 Good Excellent Good Excellent Excellent attachemnts(about 10-15 (20+ (about 10-15 (20+ (20+ (static culture) cells/bead)cells/bead) cells/bead) cells/bead) cells/bead) ESD3 Excellent ExcellentExcellent Poor at 24 h, Good attachemnt failed at 48 h (static culture)ESD3 Excellent Good Good Failed Failed attachement (spinner flask)BG01V/hOG Good Good Good Good Not tested (static culture)

All of the microcarriers tested were good or excellent with regard toHT1080 cell attachment. In contrast, ESD3 stem cells were observed toattach to only some of the microcarriers. Microcarriers that had apolypeptide density of 100 nmol/mg or more were able to supportattachment of ESD3 cells, while those having a polypeptide density of100 nmol/mg or less were not able to support attachment of ESD3 cells atall or in stirred culture conditions (spinner flask). With regard tohuman embryonic stem cells, BGO1V/hOG, attachment was good to allmicrocarriers tested. While not tested, the ability of the humanembryonic stem cells to the some of microcarriers under stirredconditions may not be as good under stirred conditions. None-the-less,the results presented herein show that a variety of microcarriers asdescribed herein support attachment of a variety of cell types.

Based on the results presented in this Example, it appears that thecrosslinker concentration and type can have an impact on the EWC andpeptide loading, which may also affect the ability of the microcarrierto support culture of various cell types. Accordingly, the nature andamount of crosslinker, as well of other monomers, can be readilymodified to tune the microcarrier to support culture for a desired celltype.

Thus, embodiments of SYNTHETIC MICROCARRIERS FOR CULTURING CELLS aredisclosed. One skilled in the art will appreciate that the arrays,compositions, kits and methods described herein can be practiced withembodiments other than those disclosed. The disclosed embodiments arepresented for purposes of illustration and not limitation.

1. A microcarrier for cell culture, comprising: a polymeric microcarrierbase formed from copolymerization of a mixture of monomers including (i)an uncharged hydrophilic unsaturated monomer having a hydroxyl groupselected from a hydrophilic (meth)acrylate monomer having a hydroxylgroup or a hydrophilic (meth)acrylamide monomer having a hydroxyl group,(ii) a hydrophilic carboxylic acid containing unsaturated monomerselected from a carboxylic acid containing (meth)acrylate monomer or acarboxylic acid containing (meth)acrylamide monomer, and (iii) a firsthydrophilic multifunctional unsaturated monomer selected from ahydrophilic multifunctional (meth)acrylate monomer or a hydrophilicmultifunctional (meth)acrylamide monomer; and a polypeptide conjugatedto the microcarrier base, wherein the microcarrier base has anequilibrium water content of greater than 75%.
 2. A microcarrieraccording to claim 1, wherein the mixture of monomers includes (i) 30 to70 parts per weight of the uncharged hydrophilic unsaturated monomerhaving a hydroxyl group; (ii) 20 to 60 parts per weight of thehydrophilic carboxylic acid containing unsaturated monomer; and (iii) 1to 15 parts by weight of the first hydrophilic multifunctionalunsaturated monomer.
 3. A microcarrier according to claim 1, wherein theuncharged hydrophilic unsaturated monomer having a hydroxyl group is amonomer according to Formula (I) or Formula (II):

where A is H or methyl, and where B is C1-C6 straight or branched chainalcohol or ether. In some embodiments, B is C1-C4 straight or branchedchain alcohol; or

where A is hydrogen or methyl, and where B is C1-C6 straight or branchedchain alcohol or ether.
 4. A microcarrier according to claim 2, whereinthe uncharged hydrophilic unsaturated monomer having a hydroxyl group isa monomer according to Formula (I) or Formula (II):

where A is H or methyl, and where B is C1-C6 straight or branched chainalcohol or ether. In some embodiments, B is C1-C4 straight or branchedchain alcohol; or

where A is hydrogen or methyl, and where B is C1-C6 straight or branchedchain alcohol or ether.
 5. A microcarrier according to claim 1, whereinthe uncharged hydrophilic unsaturated monomer having a hydroxyl group isselected from the group consisting of hydroxypropyl methacrylate,2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, glycerolmethacrylate, hydroxypropyl acrylate, 4-hydroxybutyl acrylate,N-(hydroxymethyl)acrylamide, N-[Tris(hydroxymethyl)methyl]acrylamide,3-acryloylamino-1-propanol, N-acrylamido-ethoxyethanol, andN-hydroxyethyl acrylamide.
 6. A microcarrier according to claim 2,wherein the uncharged hydrophilic unsaturated monomer having a hydroxylgroup is selected from the group consisting of hydroxypropylmethacrylate, 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate,glycerol methacrylate, hydroxypropyl acrylate, 4-hydroxybutyl acrylate,N-(hydroxymethyl)acrylamide, N-[Tris(hydroxymethyl)methyl]acrylamide,3-acryloylamino-1-propanol, N-acrylamido-ethoxyethanol, andN-hydroxyethyl acrylamide.
 7. A microcarrier according to claim 1,wherein the uncharged hydrophilic unsaturated monomer having a hydroxylgroup is 2-hydroxyethyl methacrylate.
 8. A microcarrier according toclaim 2, wherein the uncharged hydrophilic unsaturated monomer having ahydroxyl group is 2-hydroxyethyl methacrylate.
 9. A microcarrieraccording to claim 1, wherein the hydrophilic carboxylic acid containingunsaturated monomer is a monomer according to Formula (III) or Formula(IV):

where A is hydrogen or methyl, and where D is C1-C6 straight or branchedchain alkyl substituted with a carboxyl group (—COOH); or

where A is hydrogen or methyl, and where D is C1-C6 straight or branchedchain alkyl substituted with a carboxyl group (—COOH).
 10. Amicrocarrier according to claim 2, wherein the hydrophilic carboxylicacid containing unsaturated monomer is a monomer according to Formula(III) or Formula (IV):

where A is hydrogen or methyl, and where D is C1-C6 straight or branchedchain alkyl substituted with a carboxyl group (—COOH); or

where A is hydrogen or methyl, and where D is C1-C6 straight or branchedchain alkyl substituted with a carboxyl group (—COOH).
 11. Amicrocarrier according to claim 1 wherein the hydrophilic carboxylicacid containing unsaturated monomer is selected from the groupconsisting of 2-carboxyethyl methacrylate, 2-carboxyethyl acrylate,acrylic acid, methacrylic acid, 2-carboxyethyl acrylamide, andacrylamidoglycolic acid.
 12. A microcarrier according to claim 2 whereinthe hydrophilic carboxylic acid containing unsaturated monomer isselected from the group consisting of 2-carboxyethyl methacrylate,2-carboxyethyl acrylate, acrylic acid, methacrylic acid, 2-carboxyethylacrylamide, and acrylamidoglycolic acid.
 13. A microcarrier according toany of claims 1, wherein the hydrophilic carboxylic acid containingunsaturated monomer is 2-carboxyethyl methacrylate.
 14. A microcarrieraccording to any of claims 2, wherein the hydrophilic carboxylic acidcontaining unsaturated monomer is 2-carboxyethyl methacrylate.
 15. Amicrocarrier according to claims 1, wherein the first hydrophilicmultifunctional unsaturated monomer is selected from the groupconsisting of N,N′methylenebisacrylamide,N,N′(1,2-dihydroxyethylene)bisacrylamide, polyethylene glycoldi(meth)acrylate, triglycerol diacrylate, propylene glycol glycerolatediacrylate, trimethylolpropane ethoxylate triacrylate, and glycerol1,3-diglycerolate diacrylate.
 16. A microcarrier according to claims 2,wherein the first hydrophilic multifunctional unsaturated monomer isselected from the group consisting of N,N′methylenebisacrylamide,N,N′(1,2-dihydroxyethylene)bisacrylamide, polyethylene glycoldi(meth)acrylate, triglycerol diacrylate, propylene glycol glycerolatediacrylate, trimethylolpropane ethoxylate triacrylate, and glycerol1,3-diglycerolate diacrylate.
 17. A microcarrier according to claim 1,wherein the first hydrophilic multifunctional unsaturated monomer isselected from the group consisting of methylene bisacrylamide anddihydroxyethylene bisacrylamide.
 18. A microcarrier according to claim1, wherein the mixture of monomers further comprises a secondmultifunctional unsaturated monomer selected from a multifunctionalunsaturated (meth)acrylate monomer or a multifunctional unsaturated(meth)acrylamide monomer.
 19. A microcarrier according to claim 18,wherein the second multifunctional unsaturated monomer is lesshydrophilic than the first hydrophilic multifunctional unsaturatedmonomer.
 20. A microcarrier according to claim 19, wherein the secondmultifunctional unsaturated monomer is tetra(ethylene glycol)dimethacrylate.
 21. A microcarrier according to claim 18, wherein thefirst hydrophilic multifunctional unsaturated monomer and the secondmultifunctional unsaturated monomer together do not exceed 15 parts byweight of the mixture of monomers.
 22. A method according to claim 21,wherein the mixture of monomers further comprises tetra(ethyleneglycol)dimethacrylate, and wherein the combination of the tetra(ethyleneglycol)dimethacrylate and the methylene bisacrylamide or thetetra(ethylene glycol)dimethacrylate and the dihydroxyethylenebisacrylamide does not exceed 15 parts by weight of the mixture ofmonomers.
 23. A microcarrier according to claim 1, wherein thepolypeptide is an RGD-containing polypeptide.
 24. A microcarrieraccording to claim 1, wherein the microcarrier has greater than 100 nmolconjugated polypeptide per milligram of microcarrier base.
 25. A methodfor culturing human embryonic stem cells comprising: contacting thecells with a cell culture medium having microcarriers according to claim1; and culturing the cells in the medium.
 26. A method for culturinghuman embryonic stem cells comprising: contacting the cells with a cellculture medium having microcarriers according to claim 20, wherein theuncharged hydrophilic unsaturated monomer having a hydroxyl group isselected from the group consisting of hydroxypropyl methacrylate,2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, glycerolmethacrylate, hydroxypropyl acrylate, 4-hydroxybutyl acrylate,N-(hydroxymethyl)acrylamide, N-[Tris(hydroxymethyl)methyl]acrylamide,3-acryloylamino-1-propanol, N-acrylamido-ethoxyethanol, andN-hydroxyethyl acrylamide and wherein the hydrophilic carboxylic acidcontaining unsaturated monomer is selected from the group consisting of2-carboxyethyl methacrylate, 2-carboxyethyl acrylate, acrylic acid,methacrylic acid, 2-carboxyethyl acrylamide, and acrylamidoglycolicacid; and culturing the cells in the medium.
 27. A method according toclaim 25, wherein the microcarrier base is formed from a mixture ofmonomers comprising is 2-hydroxyethyl methacrylate, 2-carboxyethylmethacrylate, and (i) methylene bisacrylamide or (ii) dihydroxyethylenebisacrylamide, wherein the mixture of monomers includes (i) 30 to 70parts per weight of the 2-hydroxyethyl methacrylate; (ii) 20 to 60 partsper weight of the 2-carboxyethyl methacrylate; and (iii) 1 to 15 partsby weight of methylene bisacrylamide or dihydroxyethylene bisacrylamide,and wherein the polypeptide is an RGD-containing polypeptide.